Analogs of Nitrofuran Antibiotics to Combat Resistance

ABSTRACT

Novel compounds and methods of killing or inhibiting the growth of bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 63/048,017, filed Jul. 3, 2020, the contents of which are herebyincorporated by reference in its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under GM120350 awardedby National Institutes of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

The present disclosure relates to certain compounds and methods ofkilling or inhibiting the growth of bacteria. Specifically, thecompounds target GroEL/ES protein complex.

BACKGROUND

The rise of antibiotic-resistant bacteria has become a significantthreat to human health worldwide. This has been recently re-emphasizedby the Centers for Disease Control and Prevention (CDC) in their 2019report titled “Antibiotic Resistance Threats in the United States”. Inthe US alone, more than 2.8 million infections by antibiotic resistantbacteria occur annually, with 35,000 deaths. Of particular prominence isa subset of bacteria referred to as the ESKAPE pathogens—an acronym thatrepresents Gram-positive Enterococcus faecium and Staphylococcus aureusbacteria, and Gram-negative Klebsiella pneumonia, Acinetobacterbaumannii, Pseudomonas aeruginosa, and Enterobacter species. Whileseveral classes of antibiotics that target diverse biological pathwayshave been successfully used to treat these bacteria for decades, we arein an era where derivatizing analogs to circumvent bacterial resistancehas led to diminishing returns for developing effective new clinicalcandidates. In some instances, bacterial strains have emerged that areresistant to all contemporary antibacterials, as well as older, moretoxic drugs that clinicians are reverting to in more desperatesituations (e.g. polymyxins). To counter the diminishing antibacterialpipeline, it is crucial that new antibacterial candidates are developedthat function through unexploited biological pathways. Furthermore, ofparticular urgency is to identify new antibiotic candidates that areeffective against Gram-negative bacteria, since their lipopolysaccharide(LPS) outer membranes and efficient efflux pumps make them highlyimpermeable and intrinsically resistant to many antibacterial agents.

To circumvent pre-disposed resistance mechanisms, there is a need fornew antibacterials that function through new mechanisms of action andagainst previously unexploited pathways.

SUMMARY

In one aspect, the disclosure relates to a compound of the formula I

wherein

R¹ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl, wherein eachhydrogen atom in C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl isoptionally substituted with halogen, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or—N(R⁵)SO₂R⁶;

R² is hydrogen;

or R¹ and R² combine with the atoms to which they are attached to form a3- to 7-membered heterocycloalkyl optionally substituted by oxo;

R³ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl, wherein eachhydrogen atom in C₆-C₁₀ aryl, biaryl, and heteroaryl is optionallysubstituted by —OR⁵ or nitro;

R⁴ is hydrogen;

R⁵ and R⁶ are each individually hydrogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl, wherein each hydrogen atom in C₁-C₆ alkyl,aryl, and heteroaryl is optionally substituted with halogen or —OC₁-C₆alkyl;

or a pharmaceutically acceptable salt thereof.

In some aspects, the compound is not

In another aspect, the disclosure relates to a compound or apharmaceutically acceptable salt thereof, having the formula II

In another aspect, the disclosure relates to a compound or apharmaceutically acceptable salt thereof, having the formula III

wherein Z is CH or N.

Additional embodiments, features, and advantages of the disclosure willbe apparent from the following detailed description and through practiceof the disclosure. The compounds of the present disclosure can bedescribed as embodiments in any of the following enumerated clauses. Itwill be understood that any of the embodiments described herein can beused in connection with any other embodiments described herein to theextent that the embodiments do not contradict one another.

1. A compound of formula (I):

wherein

R¹ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl, wherein eachhydrogen atom in C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl isoptionally substituted with halogen, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or—N(R⁵)SO₂R⁶;

R² is hydrogen;

or R¹ and R² combine with the atoms to which they are attached to form a3- to 7-membered heterocycloalkyl optionally substituted by oxo;

R³ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl, wherein eachhydrogen atom in C₆-C₁₀ aryl aryl, biaryl, and mono- or bicyclicheteroaryl is optionally substituted by —OR⁵ or nitro;

R⁴ is hydrogen;

R⁵ and R⁶ are each individually hydrogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl, wherein each hydrogen atom in C₁-C₆ alkyl,C₆-C₁₀ aryl, and mono- or bicyclic heteroaryl is optionally substitutedwith halogen or —OC₁-C₆ alkyl;

or a pharmaceutically acceptable salt thereof,

provided the compound is not

2. The compound of clause 1, or a pharmaceutically acceptable saltthereof, wherein R³ is mono- or bicyclic heteroaryl optionallysubstituted by nitro.

3. The compound of clause 1, or a pharmaceutically acceptable saltthereof, wherein R³ is quinolyl optionally substituted by —OR⁵.

4. The compound of clause 1, or a pharmaceutically acceptable saltthereof, wherein R³ is napthyl optionally substituted by —OR⁵.

5. The compound of clause 1, or a pharmaceutically acceptable saltthereof, wherein R³ is furanyl optionally substituted by nitro.

6. The compound of clause 1, having the formula (II)

or a pharmaceutically acceptable salt thereof.

7. The compound of clause 1, having the formula (III)

wherein Z is CH or N;

or a pharmaceutically acceptable salt thereof.

8. The compound of any of the preceding clauses, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is monocylic heteroaryl optionallysubstituted with halogen.

9. The compound of any one of clauses 1-7, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is C₆-C₁₀ aryl optionallysubstituted with halo, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or —N(R⁵)SO₂R⁶.

10. The compound of clause 7, wherein R¹ is C₆-C₁₀ aryl optionallysubstituted by —OH, —OC₁-C₆ alkyl, O-aryl, —N(C₁-C₆ alkyl)₂,—S(O)₂N(C₁-C₆ alkyl)₂, —N(H)S(O)₂—C₆-C₁₀ aryl, —N(H)S(O)₂-monocylicheteroaryl, or —N(H)S(O)₂-bicylic heteroaryl, wherein each hydrogen atomin C₆-C₁₀aryl, or mono- or bicyclic heteroaryl is optionally substitutedwith halogen or —OC₁-C₆ alkyl.

11. The compound of any one of clauses 1-5, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is biaryl.

12. The compound of clause 1, or a pharmaceutically acceptable saltthereof, selected from the group consisting of

13. The compound of clause 12, or a pharmaceutically acceptable saltthereof, selected from the group consisting of

14. The compound of clause 11, or a pharmaceutically acceptable saltthereof, selected from the group consisting of

15. The compound of clause 10, or a pharmaceutically acceptable saltthereof, selected from the group consisting of

16. The compound of clause 13, or a pharmaceutically acceptable saltthereof, selected from the group consisting of

17. A pharmaceutical composition comprising a compound of any one of thepreceding clauses, or a pharmaceutically acceptable salt thereof, andoptionally at least one diluent, carrier or excipient.

18. A method of treating a bacterial infection comprising administeringto a subject in need of such treatment an effective amount of at leastone compound of any one of clauses 1 to 16, or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are graphs showing correlation plots of IC₅₀ valuesfor nitrofuran analogs and hydroxyquinoline analogs evaluated in therespective biochemical assays, wherein FIG. 1A shows the inhibitorpotencies in the GroEL/ES-dMDH and the GroEL/ES-dRho refolding assays,FIG. 1B shows the inhibitor potencies in the native Rho and the nativeMDH assays, and FIG. 1C shows the inhibitor potencies in theGroEL/ES-dMDH refolding assay with and without the E. coli NfsBnitroreductase.

FIGS. 2A, 2B, and 2C are graphs showing the correlation plots ofinhibitor potency of hydroxyquinoline analogs and nitrofuran analogs inthe in situ NfsB-GroEL/ES-dMDH refolding assay and in the presence ofvarious bacterium, wherein FIG. 2A shows the inhibition of E. faecium,FIG. 2B shows the inhibition of S. aureus, and FIG. 2C shows theinhibition of E. coli.

FIGS. 3A, 3B, and 3C are graphs showing correlation plots examining theselectivity of hydroxyquinoline analogs and nitrofuran analogsinhibiting the proliferation of various bacterium over the cytotoxicityto human FHs 74 Int small intestine cells, wherein FIG. 3A shows theinhibition of E. faecium, FIG. 3B shows the inhibition of S. aureus, andFIG. 3C shows the inhibition of E. coli.

FIGS. 4A, 4B, and 4C are graphs showing the ability of E. coli togenerate resistance to various inhibitors over time, wherein FIG. 4Ashows the resistance to nifuroxazide, FIG. 4B shows resistance tonitrofurantoin, and FIG. 4C shows the resistance to compound 17.

FIGS. 5A, 5B, and 5C are graphs showing dose-response curves for variousinhibitors tested against the susceptible parent E. coli (whitetriangle), the maximally-resistant strain of E. coli developed to therespective inhibitor (black triangle), and follow-up proliferationassays for the resistant strain of E. coli tested after serial passagingin the absence of the inhibitor (grey triangle), wherein FIG. 5A showsthe curve for nifuroxazide, FIG. 5B shows the curve for nitrofurantoin,and FIG. 5C shows the curve for compound 17.

FIGS. 6A, 6B, and 6C are graphs showing dose-responsive curves forvarious inhibitors tested against resistant E. coli strains, whereinFIG. 6A shows the curve for nifuroxazide resistant E. coli, FIG. 6Bshows the curve for nitrofurantoin resistant E. coli, and FIG. 6C showsthe curve for compound 17 resistant E. coli.

FIGS. 7A, 7B, and 7C are graphs showing correlation plots examining theselectivity of hydroxyquinoline analogs and nitrofuran analogsinhibiting the proliferation of various bacterium over the cytotoxicityto human FHC colon cells, wherein FIG. 7A shows the inhibition of E.faecium, FIG. 7B shows the inhibition of S. aureus, and FIG. 7C showsthe inhibition of E. coli.

FIGS. 8A, 8B, and 8C are graphs showing dose-response curves for variousinhibitors tested against the susceptible parent E. coli (whitetriangle), the maximally-resistant strain of E. coli developed to therespective inhibitor (black triangle), and follow-up proliferationassays for the resistant strain of E. coli tested after serial passagingin the absence of the inhibitor (grey triangle), wherein FIG. 8A showsthe curve for nifuroxazide, FIG. 8B shows the curve for nitrofurantoin,and FIG. 8C shows the curve for compound 17.

FIGS. 9A, 9B, and 9C are graphs showing dose-responsive curves forvarious inhibitors tested against resistant E. coli strains, whereinFIG. 9A shows the curve for nifuroxazide resistant E. coli, FIG. 9Bshows the curve for nitrofurantoin resistant E. coli, and FIG. 9C showsthe curve for compound 17 resistant E. coli.

FIG. 10 shows an illustration of a molecular pathway.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications, and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

Chemical nomenclature for compounds described herein has generally beenderived using the commercially-available ACD/Name 2014 (ACD/Labs) orChemBioDraw Ultra 19.1 (Perkin Elmer).

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentdisclosure and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

Definitions

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched and contains from 1 to 20 carbon atoms. It is tobe further understood that in certain embodiments, alkyl may beadvantageously of limited length, including C₁-C₁₂, C₁-C₁₀, C₁-C₉,C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄. Illustratively, such particularlylimited length alkyl groups, including C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄,and the like may be referred to as “lower alkyl.” Illustrative alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,3-pentyl, neopentyl, hexyl, heptyl, octyl, and the like. Alkyl may besubstituted or unsubstituted. Typical substituent groups includecycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halo, carbonyl, oxo, (═O),thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, N-sulfamido, C-carboxy, O-carboxy, nitro, and amino,or as described in the various embodiments provided herein. It will beunderstood that “alkyl” may be combined with other groups, such as thoseprovided above, to form a functionalized alkyl. By way of example, thecombination of an “alkyl” group, as described herein, with a “carboxy”group may be referred to as a “carboxyalkyl” group. Other non-limitingexamples include hydroxyalkyl, aminoalkyl, and the like.

As used herein, the term “aryl” refers to an all-carbon monocyclic orfused-ring polycyclic groups of 6 to 12 carbon atoms having a completelyconjugated pi-electron system. It will be understood that in certainembodiments, aryl may be advantageously of limited size such as C₆-C₁₀aryl. Illustrative aryl groups include, but are not limited to, phenyl,naphthylenyl and anthracenyl. The aryl group may be a biaryl groupcontaining two fused rings, for example napthyl. The aryl group may beunsubstituted, or substituted as described for alkyl or as described inthe various embodiments provided herein.

As used herein, the term “heterocycloalkyl” refers to a monocyclic orfused ring group having in the ring(s) from 3 to 12 ring atoms, in whichat least one ring atom is a heteroatom, such as nitrogen, oxygen orsulfur, the remaining ring atoms being carbon atoms. Heterocycloalkylmay optionally contain 1, 2, 3 or 4 heteroatoms. A heterocycloalkylgroup may be fused to another group such as another heterocycloalkyl ora heteroaryl group. Heterocycloalkyl may also have one of more doublebonds, including double bonds to nitrogen (e.g., C═N or N═N) but doesnot contain a completely conjugated pi-electron system. It will beunderstood that in certain embodiments, heterocycloalkyl may beadvantageously of limited size such as 3- to 7-memberedheterocycloalkyl, 5- to 7-membered heterocycloalkyl, 3-, 4-, 5- or6-membered heterocycloalkyl, and the like. Heterocycloalkyl may beunsubstituted, or substituted as described for alkyl or as described inthe various embodiments provided herein. Illustrative heterocycloalkylgroups include, but are not limited to, oxiranyl, thianaryl, azetidinyl,oxetanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl,piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, piperazinyl,oxepanyl, 3,4-dihydro-2H-pyranyl, 5,6-dihydro-2H-pyranyl, 2H-pyranyl, 1,2, 3, 4-tetrahydropyridinyl, and the like. Illustrative examples ofheterocycloalkyl groups shown in graphical representations include thefollowing entities, in the form of properly bonded moieties:

As used herein, the term “heteroaryl” refers to a monocyclic or fusedring group of 5 to 12 ring atoms containing one, two, three or four ringheteroatoms selected from nitrogen, oxygen and sulfur, the remainingring atoms being carbon atoms, and also having a completely conjugatedpi-electron system. It will be understood that in certain embodiments,heteroaryl may be advantageously of limited size such as 3- to7-membered heteroaryl, 5- to 7-membered heteroaryl, and the like.Heteroaryl may be unsubstituted, or substituted as described for alkylor as described in the various embodiments provided herein. Illustrativeheteroaryl groups include, but are not limited to, pyrrolyl, furanyl,thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl,pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl,pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl,isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl andcarbazoloyl, and the like. Illustrative examples of heteroaryl groupsshown in graphical representations, include the following entities, inthe form of properly bonded moieties:

As used herein, “hydroxy” or “hydroxyl” refers to an —OH group.

As used herein, “alkoxy” refers to both an —O-(alkyl) or an—O-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, methoxy, ethoxy, propoxy, butoxy,cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and thelike.

The term “oxo” represents a carbonyl oxygen. For example, a cyclopentylsubstituted with oxo is cyclopentanone.

As used herein, “halogen” refers to fluorine, chlorine, bromine, oridodine.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance may but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “wherein each hydrogenatom in C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl isoptionally substituted with halogen” means that a halogen may be butneed not be present on any of the C₆-C₁₀ aryl, biaryl, or mono- orbicyclic heteroaryl by replacement of a hydrogen atom for each halogengroup, and the description includes situations where the C₆-C₁₀ aryl,biaryl, or mono- or bicyclic heteroaryl is substituted with a halogengroup and situations where C₆-C₁₀ aryl, biaryl, or mono- or bicyclicheteroaryl is not substituted with the halogen group.

As used herein, “independently” means that the subsequently describedevent or circumstance is to be read on its own relative to other similarevents or circumstances. For example, in a circumstance where severalequivalent hydrogen groups are optionally substituted by another groupdescribed in the circumstance, the use of “independently optionally”means that each instance of a hydrogen atom on the group may besubstituted by another group, where the groups replacing each of thehydrogen atoms may be the same or different. Or for example, wheremultiple groups exist all of which can be selected from a set ofpossibilities, the use of “independently” means that each of the groupscan be selected from the set of possibilities separate from any othergroup, and the groups selected in the circumstance may be the same ordifferent.

As used herein, the phrase “R¹ and R² combine with the atoms to whichthey are attached to form a 3- to 7-membered heterocycloalkyl optionallysubstituted by oxo” also means that R¹ and R² are taken together withthe carbon or nitrogen atoms to which they are attached to form a a 3-to 7-membered heterocycloalkyl that is optionally substituted. Inparticular, “R¹ and R² combine with the atoms to which they are attachedto form a 3- to 7-membered heterocycloalkyl optionally substituted byoxo:

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts with counter ions which may be used in pharmaceuticals. See,generally, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977, 66, 1-19. Preferred pharmaceutically acceptable salts are thosethat are pharmacologically effective and suitable for contact with thetissues of subjects without undue toxicity, irritation, or allergicresponse. A compound described herein may possess a sufficiently acidicgroup, a sufficiently basic group, both types of functional groups, ormore than one of each type, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt. Such salts include:

(1) acid addition salts, which can be obtained by reaction of the freebase of the parent compound with inorganic acids such as hydrochloricacid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, andperchloric acid and the like, or with organic acids such as acetic acid,oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaricacid, citric acid, succinic acid or malonic acid and the like; or

(2) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,trimethamine, N-methylglucamine, and the like.

Pharmaceutically acceptable salts are well known to those skilled in theart, and any such pharmaceutically acceptable salt may be contemplatedin connection with the embodiments described herein. Examples ofpharmaceutically acceptable salts include sulfates, pyrosulfates,bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides,bromides, iodides, acetates, propionates, decanoates, caprylates,acrylates, formates, isobutyrates, caproates, heptanoates, propiolates,oxalates, malonates, succinates, suberates, sebacates, fumarates,maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates,chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates,methoxybenzoates, phthalates, sulfonates, methylsulfonates,propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, and mandelates. Lists of other suitable pharmaceuticallyacceptable salts are found in Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Company, Easton, Pa., 1985.

The present disclosure also relates to pharmaceutically activemetabolites of compounds of Formula I, II, or III and uses of suchmetabolites in the methods of the disclosure. A “pharmaceutically activemetabolite” means a pharmacologically active product of metabolism inthe body of a compound of Formula I, II, or III, or salt thereof.Prodrugs and active metabolites of a compound may be determined usingroutine techniques known or available in the art. See, e.g., Bertoliniet al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci.1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230;Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design of Prodrugs(Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs,Drug Design and Development (Krogsgaard-Larsen et al., eds., HarwoodAcademic Publishers, 1991).

Any formula depicted herein is intended to represent a compound of thatstructural formula as well as certain variations or forms. For example,a formula given herein is intended to include a racemic form, or one ormore enantiomeric, diastereomeric, or geometric isomers, or a mixturethereof. Additionally, any formula given herein is intended to referalso to a hydrate, solvate, or polymorph of such a compound, or amixture thereof. For example, it will be appreciated that compoundsdepicted by a structural formula containing the symbol “

” include both stereoisomers for the carbon atom to which the symbol “

” is attached, specifically both the bonds “

” and “

” are encompassed by the meaning of “

”.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the disclosure include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O ³¹P, ³²P, ³⁵S, ¹⁸F,³⁶Cl, and ¹²⁵I, respectively. Such isotopically labelled compounds areuseful in metabolic studies (preferably with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques[such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT)] including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Further,substitution with heavier isotopes such as deuterium (i.e., ²H) mayafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements. Isotopically labeled compounds of this disclosure andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Any disubstituent referred to herein is meant to encompass the variousattachment possibilities when more than one of such possibilities areallowed. For example, reference to disubstituent -A-B-, where A≠B,refers herein to such disubstituent with A attached to a firstsubstituted member and B attached to a second substituted member, and italso refers to such disubstituent with A attached to the secondsubstituted member and B attached to the first substituted member.

REPRESENTATIVE EMBODIMENTS

Compounds described herein may be of the formula I

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound may be of formula II

or a pharmaceutically acceptable salt thereof. In still otherembodiments, the compound is of formula III

wherein Z is CH or N;

or a pharmaceutically acceptable salt thereof.

In illustrative embodiments, compounds of formula I do not include

In some embodiments, R¹ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclicheteroaryl. In some embodiments, R¹ is C₆-C₁₀ aryl or biaryl. In someembodiments, R¹ is C₆-C₁₀ aryl, biaryl, or monocyclic heteroaryl. Insome embodiments, R¹ is C₆-C₁₀ aryl. In some embodiments, R¹ is C₆-C₁₀biaryl. In some embodiments, R¹ is monocyclic heteroaryl. In someembodiments, R¹ is bicyclic heteroaryl. Illustratively, each hydrogenatom in C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl of R¹ isoptionally substituted with halogen, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or—N(R⁵)SO₂R⁶. In some embodiments, R¹ is monocylic heteroaryl optionallysubstituted with halogen. In some embodiments, R¹ is C₆-C₁₀ aryloptionally substituted with halo, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or—N(R⁵)SO₂R⁶. In some embodiments, R¹ is C₆-C₁₀ aryl optionallysubstituted by —OH, —OC₁-C₆ alkyl, O-aryl, —N(C₁-C₆ alkyl)₂,—S(O)₂N(C₁-C₆ alkyl)₂, —N(H)S(O)₂—C₆-C₁₀ aryl, —N(H)S(O)₂-monocylicheteroaryl, or —N(H)S(O)₂-bicylic heteroaryl, wherein each hydrogen atomin C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl is optionallysubstituted with halogen or —OC₁-C₆ alkyl. In some embodiments, acompound of formula I or a pharmaceutically acceptable salt thereof,wherein R¹ is biaryl.

In some embodiments, R² is hydrogen.

In still some embodiments, R¹ and R² combine with the atoms to whichthey are attached to form a 3- to 7-membered heterocycloalkyl. In someembodiments, the 3- to 7-membered heterocycloalkyl is substituted byoxo.

In some embodiments, R³ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclicheteroaryl. In some embodiments, R³ is C₆-C₁₀ aryl, biaryl, ormonocyclic heteroaryl. In some embodiments, R³ is C₆-C₁₀ aryl or biaryl.In some embodiments, R³ is C₆-C₁₀ aryl. In some embodiments, R³ isC₆-C₁₀ biaryl. In some embodiments, R³ is monocyclic heteroaryl. In someembodiments, R³ is bicyclic heteroaryl.

In some embodiments, wherein R³ is C₆-C₁₀ aryl, biaryl, or mono- orbicyclic heteroaryl, each hydrogen atom in aryl, biaryl, and heteroarylis optionally substituted by —OR⁵ or nitro. In some embodiments, eachhydrogen atom in aryl, biaryl, and heteroaryl of R³ is optionallysubstituted by —OR⁵. In some embodiments, each hydrogen atom in aryl,biaryl, and heteroaryl of R³ is optionally substituted by nitro. In someembodiments, R³ is heteroaryl optionally substituted by nitro. In someembodiments, R³ is quinolyl optionally substituted by —OR⁵. In someembodiments, R³ is furanyl optionally substituted by nitro.

In some embodiments, R⁴ is hydrogen.

In some embodiments, R⁵ and R⁶ are each individually hydrogen, C₁-C₆alkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl, wherein eachhydrogen atom in C₁-C₆ alkyl, aryl, and heteroaryl is optionallysubstituted with halogen or —OC₁-C₆ alkyl.

In some embodiments, R⁵ is a hydrogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl, wherein each hydrogen atom in C₁-C₆ alkyl,aryl, and heteroaryl is optionally substituted with halogen or —OC₁-C₆alkyl. In some embodiments, R⁵ is a hydrogen. In some embodiments, R⁵ isa C₁-C₆ alkyl. In some embodiments, each hydrogen atom in C₁-C₆ alkyl isoptionally substituted with halogen or —OC₁-C₆ alkyl.

In some embodiments, R⁵ is C₆-C₁₀ aryl. In some embodiments, eachhydrogen atom in the aryl is optionally substituted with halogen or—OC₁-C₆ alkyl. In some embodiments, R⁵ is monocyclic heteroaryl. In someembodiments, each hydrogen of monocyclic heteroaryl is optionallysubstituted with halogen or —OC₁-C₆ alkyl. In some embodiments, R⁵ is abicyclic heteroaryl. In some embodiments, each hydrogen atom of bicyclicheteroaryl is optionally substituted with halogen or —OC₁-C₆ alkyl.

In some embodiments, R⁶ is a hydrogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl, wherein each hydrogen atom in C₁-C₆ alkyl,aryl, and heteroaryl is optionally substituted with halogen or —OC₁-C₆alkyl. In some embodiments, R⁶ is a hydrogen. In some embodiments, R⁶ isa C₁-C₆ alkyl. In some embodiments, each hydrogen atom in C₁-C₆ alkyl isoptionally substituted with halogen or —OC₁-C₆ alkyl.

In some embodiments, R⁶ is C₆-C₁₀ aryl. In some embodiments, eachhydrogen atom in the aryl is optionally substituted with halogen or—OC₁-C₆ alkyl. In some embodiments, R⁶ is monocyclic heteroaryl. In someembodiments, each hydrogen of monocyclic heteroaryl is optionallysubstituted with halogen or —OC₁-C₆ alkyl. In some embodiments, R⁶ is abicyclic heteroaryl. In some embodiments, each hydrogen atom of bicyclicheteroaryl is optionally substituted with halogen or —OC₁-C₆ alkyl.

In some embodiments, the compound of formula I having the formula (II)

or a pharmaceutically acceptable salt thereof. In some embodiments, R¹is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl. In someembodiments, R¹ is C₆-C₁₀ aryl, biaryl, or monocyclic heteroaryl. Insome embodiments, R¹ is C₆-C₁₀ aryl or biaryl. In some embodiments, R¹is C₆-C₁₀ aryl. In some embodiments, R¹ is C₆-C₁₀ biaryl. In someembodiments, R¹ is monocyclic heteroaryl. In some embodiments, R¹ isbicyclic heteroaryl. In some embodiments, each hydrogen atom in C₆-C₁₀aryl, biaryl, or mono- or bicyclic heteroaryl of R¹ is optionallysubstituted with halogen, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or —NR⁵SO₂R⁶.

In some embodiments, R² is hydrogen. In some embodiments, R¹ and R²combine with the atoms to which they are attached to form a 3- to7-membered heterocycloalkyl. In some embodiments, the 3- to 7-memberedheterocycloalkyl is optionally substituted by oxo.

In some embodiments, R⁴ is hydrogen.

In some embodiments, the compound of formula I, having the formula (III)

or a pharmaceutically acceptable salt thereof.

In some embodiments, wherein Z is CH or N;

In some embodiments, the compound is of formula I, formula II, orformula III or a pharmaceutically acceptable salt thereof, wherein R¹ ismonocylic heteroaryl optionally substituted with halogen. In someembodiments, the compound is of formula I, formula II, or formula III ora pharmaceutically acceptable salt thereof, wherein R¹ is C₆-C₁₀ aryloptionally substituted with halo, —OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or—NR⁵SO₂R⁶.

In some embodiments, wherein the compound is formula III, wherein R¹ isC₆-C₁₀ aryl optionally substituted by —OH, —OC₁-C₆ alkyl, O-aryl,—N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —NHSO₂-aryl, —NHSO₂-heteroaryl.In some embodiments, the aryl is optionally substituted with halogen or—OC₁-C₆ alkyl.

In some embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of

In some embodiments, the compound of formula III, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of

In some embodiments, the compound of clause 11, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of

In some embodiments, the compound of clause 10, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of

In some embodiments, the compound of clause 13, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of

A pharmaceutical composition comprising a compound of formula I, formulaII, formula III, or a pharmaceutically acceptable salt thereof, andoptionally at least one diluent, carrier or excipient.

A method of treating a bacterial infection comprising administering to asubject in need of such treatment an effective amount of at least onecompound of formula I, formula II, formula III, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, a method of killing or inhibiting the growth ofbacteria is provided. The method comprises contacting the bacteria witha compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of inhibiting the GroEL/ES protein complexin a bacterium comprising contacting the bacterium with a compound offormula I, formula II, formula III, or a pharmaceutical acceptable saltthereof.

In some embodiments, a metabolite of formula I, formula II, formula III,or a pharmaceutically acceptable salt. In some embodiments, a method ofinhibiting the growth of a bacterium with the metabolite of formula I,formula II, or formula III is provide comprising contacting thebacterium with a compound to the bacterium, wherein the bacteriametabolizes the compound to the metabolite.

In some embodiments, for any of the methods described, the bacterium isgram negative or gram positive. In some embodiments, the bacterium isgram negative. In some embodiments, the bacterium is gram positive.

The following represent illustrative embodiments of compounds of theformula I:

Compound Structure Name  1

N′-((8-hydroxyquinolin-5-yl)methylene)-4- methoxybenzohydrazide.  2

1-(((8-hydroxyquinolin-5- yl)methylene)amino)imidazolidine-2,4-dione  3

N′-((8-hydroxyquinolin-5- yl)methylene)thiophene-2-carbohydrazide  4

5-chloro-N′-((8-hydroxyquinolin-5-yl)methylene)thiophene-2-carbohydrazide  5

N′-((8-hydroxyquinolin-5- yl)methylene)benzohydrazide  6

N′-((8-hydroxyquinolin-5- yl)methylene)isonicotinohydrazide  7

4-hydroxy-N′-((8-hydroxyquinolin-5- yl)methylene)benzohydrazide  8

4-(dimethylamino)-N′-((8-hydroxyquinolin- 5-yl)methylene)benzohydrazide 9

N′-((8-hydroxyquinolin-5-yl)methylene)-1- naphthohydrazide 10

N′-((8-hydroxyquinolin-5-yl)methylene)-2- phenoxybenzohydrazide 11

N′-((8-hydroxyquinolin-5-yl)methylene)-3- phenoxybenzohydrazide 12

N,N-diethyl-3-(2-((8-hydroxyquinolin-5- yl)methylene)hydrazine-1-carbonyl)benzenesulfonamide 13

N-(4-(2-((8-hydroxyquinolin-5- yl)methylene)hydrazine-1-carbonyl)phenyl)thiophene-2-sulfonamide 14

4-bromo-N-(4-(2-((8-hydroxyquinolin-5- yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide 15

4-ethoxy-N-(4-(2-((8-hydroxyquinolin-5- yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide 16

N′-((5-nitrofuran-2-yl)methylene)thiophene- 2-carbohydrazide 17

5-chloro-N′-((5-nitrofuran-2- yl)methylene)thiophene-2-carbohydrazide 18

N′-((5-nitrofuran-2- yl)methylene)benzohydrazide 19

N′-((5-nitrofuran-2- yl)methylene)isonicotinohydrazide 20

4-methoxy-N′-((5-nitrofuran-2- yl)methylene)benzohydrazide 21

4-(dimethylamino)-N′-((5-nitrofuran-2- yl)methylene)benzohydrazide 22

N′-((5-nitrofuran-2-yl)methylene)-1- naphthohydrazide 23

N′-((5-nitrofuran-2-yl)methylene)-2- phenoxybenzohydrazide 24

N′-((5-nitrofuran-2-yl)methylene)-3- phenoxybenzohydrazide 25

N,N-diethyl-3-(2-((5-nitrofuran-2- yl)methylene)hydrazine-1-carbonyl)benzenesulfonamide 26

N-(4-(2-((5-nitrofuran-2- yl)methylene)hydrazine-1-carbonyl)phenyl)thiophene-2-sulfonamide 27

4-bromo-N-(4-(2-((5-nitrofuran-2- yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide 28

4-ethoxy-N-(4-(2-((5-nitrofuran-2- yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide 29

N′-((4-hydroxynaphthalen-1-yl)methylene)- 4-methoxybenzohydrazide 30

N′-((4-hydroxynaphthalen-1- yl)methylene)benzohydrazide 31

4-methoxy-N′-(quinolin-5- ylmethylene)benzohydrazide 32

N′-(quinolin-5-ylmethylene)benzohydrazide 33

4-methoxy-N′-(naphthalen-1- ylmethylene)benzohydrazide 34

N′-(naphthalen-1- ylmelhylene)benzohydrazide 35

N′-(4-hydroxybenzylidene)-4- methoxybenzohydrazide 36

N′-(4-hydroxybenzylidene) benzohydrazide 37

N′-benzylidene-4- methoxybenzohydrazide 38

N′-benzylidenebenzohydrazide 39

N′-(furan-2-ylmethylene)-4- methoxybenzohydrazide 40

Nitroxoline 41

Nifuroxazide 42

Nitrofurantoin

CHEMICAL SYNTHESIS

Exemplary chemical entities useful in methods of the description willnow be described by reference to illustrative synthetic schemes fortheir general preparation below and the specific examples that follow.Artisans will recognize that, to obtain the various compounds herein,starting materials may be suitably selected so that the ultimatelydesired substituents will be carried through the reaction scheme with orwithout protection as appropriate to yield the desired product.Alternatively, it may be necessary or desirable to employ, in the placeof the ultimately desired substituent, a suitable group that may becarried through the reaction scheme and replaced as appropriate with thedesired substituent. Furthermore, one of skill in the art will recognizethat the transformations shown in the schemes below may be performed inany order that is compatible with the functionality of the particularpendant groups.

Abbreviations: The examples described herein use materials, includingbut not limited to, those described by the following abbreviations knownto those skilled in the art:

g grams mmol millimoles mL milliliters EtOAc ethyl acetate MHz megahertzppm parts per million Å angstrom δ chemical shift s singlet d doublet brbroad m multiplet Hz hertz ° C. degrees Celsius J coupling constant_(d6-)DMSO deuterated dimethyl sulfoxide min minutes hr (or h) hours Mmolar MS mass spectrometry m/z mass-to-charge ratio TFA trifluoroaceticacid μM micromolar ATP adenosine triphosphate DCM dichloromethane MDHMalate dehydrogenase Rho Rhodanese R.T. Room temperature IC₅₀ Inhibitoryconcentration for half-maximal signal in biochemical assay EC₅₀Effective concentration for half-maximal signal in bacterialproliferation assays CC₅₀ Cytotoxicity concentration for half-maximalsignal in human cell viability assays

Example 1

General Synthetic Method. Unless otherwise stated, all chemicals werepurchased from commercial suppliers and used without furtherpurification. Reaction progress was monitored by thin-layerchromatography on silica gel 60 F254 coated glass plates (EM Sciences).Flash chromatography was performed using a Biotage Isolera One flashchromatography system and eluting through Biotage KP-Sil Zip or Snapsilica gel columns for normal-phase separations (hexanes:EtOAcgradients), or Snap KP-C18-HS columns for reverse-phase separations(H₂O:MeOH gradients). Reverse-phase high-performance liquidchromatography (RP-HPLC) was performed using a Waters 1525 binary pump,2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707autosampler. For preparatory HPLC purification, samples werechromatographically separated using a Waters XSelect CSH C18 OBD prepcolumn (part number 186005422, 130 Å pore size, 5 μm particle size,19×150 mm), eluting with a H₂O:CH₃CN gradient solvent system. Lineargradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B(A=95:5 H₂O:CH₃CN, 0.05% TFA; B=5:95 H₂O:CH₃CN, 0.05% TFA). Productsfrom normal-phase separations were concentrated directly, andreverse-phase separations were concentrated, diluted with H₂O, frozen,and lyophilized. For primary compound purity analyses (HPLC-1), sampleswere chromatographically separated using a Waters XSelect CSH C18 column(part number 186005282, 130 Å pore size, 5 μm particle size, 3.0×150mm), eluting with the above H₂O:CH₃CN gradient solvent systems. Forsecondary purity analyses (HPLC-2) of final test compounds, samples werechromatographically separated using a Waters XBridge C18 column (partnumber 186003132, 130 Å pore size, 5.0 μm particle size, 3.0×100 mm),eluting with a H₂O:MeOH gradient solvent system. Linear gradients wererun from either 100:0, 80:20, 60:40, or 20:80 A:B to 0:100 A:B (A=95:5H₂O:MeOH, 0.05% TFA; B=5:95 H₂O:MeOH, 0.05% TFA). Test compounds werefound to be >95% in purity from both RP-HPLC analyses. Mass spectrometrydata were collected using either Agilent LC 1200-MS 6130 or Agilent LC1290-MS 6545 Q-TOF analytical LC-MS instruments at the IU ChemicalGenomics Core Facility (CGCF). ¹H-NMR spectra were recorded on a Bruker300 MHz spectrometer at the IU CGCF. Chemical shifts are reported inparts per million and calibrated to the d₆-DMSO solvent peaks at 2.50ppm. A representative synthesis of all analogs is presented below in thecontext of compound 1.

Example 2

Analog 1:N′-((8-hydroxyquinolin-5-yl)methylene)-4-methoxybenzohydrazide. To astirring mixture of 4-methoxybenzoic acid hydrazide (668 mg, 4.02 mmol)and 8-hydroxyquinoline-5-carbaldehyde (583 mg, 3.37 mmol) was added acatalytic amount of HCl (0.09 mL of a 4N solution in 1,4-dioxane, 0.36mmol) in 10 mL of DMSO, then the reaction was left to stir at R.T.overnight. The following day, the reaction was diluted with distilledwater and the precipitate was filtered, rinsed with distilled water,collected, and dried to afford 1 as a yellow solid (1.07 g, 99% yield).¹H-NMR (300 MHz, d₆-DMSO) δ 11.70 (s, 1H), 10.44 (br s, 1H), 9.61 (d,J=8.6 Hz, 1H), 8.94 (d, J=2.8 Hz, 1H), 8.81 (s, 1H), 7.90-8.01 (m, 2H),7.68-7.82 (m, 2H), 7.17 (d, J=8.1 Hz, 1H), 7.04-7.13 (m, 2H), 3.85 (s,3H); MS (ESI) C₁₈H₁₆N₃O₃ [MH]⁺ m/z expected=322.1, observed=322.1;HPLC-1=99%; HPLC-2=99%.

Example 3

Analog 2:1-(((8-hydroxyquinolin-5-yl)methylene)amino)imidazolidine-2,4-dione.¹H-NMR (300 MHz, d₆-DMSO) δ 11.30 (s, 1H), 9.66 (dd, J=8.7, 1.3 Hz, 1H),8.99 (dd, J=4.4, 1.4 Hz, 1H), 8.21 (s, 1H), 7.92 (d, J=8.2 Hz, 1H), 7.86(dd, J=8.8, 4.5 Hz, 1H), 7.27 (d, J=8.1 Hz, 1H), 4.46 (s, 2H); MS (ESI)C₁₃H₁₁N₄O₃[MH]⁺ m/z expected=271.1, observed=271.0. HPLC-1=98%,HPLC-2=98%.

Example 4

Analog 3:N′-((8-hydroxyquinolin-5-yl)methylene)thiophene-2-carbohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.86 (s, 0.6H), 11.74 (s, 0.3H), 10.46 (br s,0.8H), 9.57 (d, J=8.4 Hz, 0.6H), 8.94 (dd, J=4.1, 1.4 Hz, 1.3H), 8.79(s, 0.7H), 8.71 (s, 0.3H), 7.65-8.11 (m, 4H), 7.12-7.31 (m,2H)—Putatively mixture of ˜2:1 rotamers; MS (ESI) C₁₅H₁₂N₃O₂S [MH]⁺ m/zexpected=298.1, observed=298.0; HPLC-1=99%.

Example 5

Analog 4:5-chloro-N′-((8-hydroxyquinolin-5-yl)methylene)thiophene-2-carbohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 9.55 (d, J=8.1 Hz, 0.5H), 8.93 (dd, J=4.0,1.2 Hz, 1H), 8.84 (d, J=8.6 Hz, 0.5H), 8.74 (d, J=9.4 Hz, 1H), 8.02 (d,J=8.1 Hz, 0.5H), 7.89 (d, J=4.1 Hz, 0.5H), 7.76-7.85 (m, 0.9H),7.66-7.75 (m, 1.1H), 7.12-7.32 (m, 2H)—Putatively mixture of ˜1:1rotamers; MS (ESI) C₁₅H₁₁ClN₃O₂S [MH]⁺ m/z expected=332.1,observed=332.0; HPLC-1=97%; HPLC-2=98%.

Example 6

Analog 5: N′-((8-hydroxyquinolin-5-yl)methylene)benzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 12.14 (s, 0.8H), 12.08 (s, 0.1H), 9.74 (dd, J=8.7,1.4 Hz, 0.8H), 9.00 (dd, J=4.3, 1.5 Hz, 1H), 8.82-8.90 (m, 2.8H), 8.48(s, 0.1H), 7.90-7.98 (m, 1.8H), 7.77-7.90 (m, 1.9H), 7.69-7.74 (m,0.1H), 7.49-7.57 (m, 0.2H), 7.25 (d, J=8.1 Hz, 0.9H), 7.13-7.20 (m,0.1H)—Putatively mixture of ˜9:1 rotamers; MS (ESI) C₁₆H₁₃N₄O₂ [MH]⁺ m/zexpected=293.1, observed=293.1; HPLC-1=98%, HPLC-2=>99%.

Example 7

Analog 6: N′-((8-hydroxyquinolin-5-yl)methylene)isonicotinohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.14 (s, 0.8H), 12.08 (s, 0.1H), 9.74 (dd,J=8.7, 1.4 Hz, 0.8H), 9.00 (dd, J=4.3, 1.5 Hz, 1H), 8.82-8.90 (m, 2.8H),8.48 (s, 0.1H), 7.90-7.98 (m, 1.8H), 7.77-7.90 (m, 1.9H), 7.69-7.74 (m,0.1H), 7.49-7.57 (m, 0.2H), 7.25 (d, J=8.1 Hz, 0.9H), 7.13-7.20 (m,0.1H)—Putatively mixture of ˜9:1 rotamers; MS (ESI) C₁₆H₁₃N₄O₂ [MH]⁺ m/zexpected=293.1, observed=293.1; HPLC-1=98%, HPLC-2=>99%.

Example 8

Analog 7:4-hydroxy-N′-((8-hydroxyquinolin-5-yl)methylene)benzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.61 (s, 1H), 10.22 (br s, 1H), 9.60 (d, J=8.5 Hz,1H), 8.92 (d, J=2.9 Hz, 1H), 8.80 (s, 1H), 7.79-7.91 (m, 2H), 7.65-7.78(m, 2H), 7.14 (d, J=8.0 Hz, 1H), 6.82-6.93 (m, 2H); MS (ESI) C₁₇H₁₄N₃O₃[MH]⁺ m/z expected=308.1, observed=308.0; HPLC-1=98%; HPLC-2=>99%.

Example 9

Analog 8:4-(dimethylamino)-N′-((8-hydroxyquinolin-5-yl)methylene)benzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.52 (s, 1H), 10.36 (br s, 1H), 9.60 (d,J=8.0 Hz, 1H), 8.93 (dd, J=4.1, 1.6 Hz, 1H), 8.80 (s, 1H), 7.81-7.91 (m,2H), 7.67-7.78 (m, 2H), 7.16 (d, J=8.0 Hz, 1H), 6.72-6.82 (m, 2H), 3.00(s, 6H); MS (ESI) C₁₉H₁₉N₄O₂ [MH]⁺ m/z expected=335.2, observed=335.1;HPLC-1=>99%; HPLC-2=98%.

Example 10

Analog 9: N′-((8-hydroxyquinolin-5-yl)methylene)-1-naphthohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.99 (s, 0.3H), 11.97 (s, 0.6H), 10.45 (brs, 0.9H), 9.66 (dd, J=8.7, 1.5 Hz, 0.7H), 8.96 (dd, J=4.1, 1.6 Hz,0.7H), 8.72 (s, 0.7H), 8.64-8.69 (m, 0.3H), 8.23-8.35 (m, 1.OH),7.99-8.16 (m, 2.3H), 7.87-7.93 (m, 0.3H), 7.73-7.83 (m, 2.1H), 7.49-7.68(m, 3.4H), 7.42-7.49 (m, 0.3H), 7.18 (d, J=8.1 Hz, 0.7H), 6.96-7.03 (m,0.3H), 6.75-6.84 (m, 0.3H)—Putatively mixture of ˜7:3 rotamers; MS (ESI)C₂₁H₁₀N₃O₂[MH]⁺ m/z expected=342.1, observed=342.1; HPLC-1=>99%;HPLC-2=99%.

Example 11

Analog 10:N′-((8-hydroxyquinolin-5-yl)methylene)-2-phenoxybenzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.73 (s, 0.4H), 11.70 (s, 0.6H), 10.39 (br s,0.8H), 9.57 (dd, J=8.7, 1.5 Hz, 0.6H), 8.92 (dd, J=4.1, 1.5 Hz, 0.6H),8.78-8.89 (m, 0.8H), 8.65 (s, 0.6H), 8.31 (s, 0.4H), 7.66-7.78 (m,1.8H), 7.47-7.58 (m, 1.7H), 7.35-7.45 (m, 1.3H), 7.20-7.34 (m, 1.4H),7.10-7.19 (m, 2H), 7.03-7.10 (m, 1.6H), 6.93-7.03 (m, 1.4H), 6.82-6.89(m, 0.8H)—Putatively mixture of ˜3:2 rotamers; MS (ESI) C₂₃H₁₈N₃O₃ [MH]⁺m/z expected=384.1, observed=384.2; HPLC-1=>99%; HPLC-2=99%.

Example 12

Analog 11:N′-((8-hydroxyquinolin-5-yl)methylene)-3-phenoxybenzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.83 (s, 1H), 10.46 (br s, 1H), 9.59 (dd, J=8.7,1.4 Hz, 1H), 8.94 (dd, J=4.1, 1.5 Hz, 1H), 8.81 (s, 1H), 7.68-7.82 (m,3H), 7.53-7.64 (m, 2H), 7.39-7.50 (m, 2H), 7.13-7.28 (m, 3H), 7.03-7.13(m, 2H); MS (ESI) C₂₃H₁₈N₃O₃ [MH]⁺ m/z expected=384.1, observed=384.1;HPLC-1=>99%; HPLC-2=>99%.

Example 13

Analog 12:N,N-diethyl-3-(2-((8-hydroxyquinolin-5-yl)methylene)hydrazine-1-carbonyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.06 (s, 1H), 10.50 (br s, 1H), 9.62 (dd,J=8.7, 1.5 Hz, 1H), 8.95 (dd, J=4.0, 1.5 Hz, 1H), 8.85 (s, 1H), 8.36 (s,1H), 8.24 (d, J=7.8 Hz, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.71-7.84 (m, 3H),7.18 (d, J=8.0 Hz, 1H), 3.22 (q J=7.1 Hz, 4H), 1.06 (t, J=7.1 Hz, 6H);MS (ESI) C₂₁H₂₃N₄O₄S [MH]⁺ m/z expected=427.1, observed=427.2;HPLC-1=99%; HPLC-2=99%.

Example 14

Analog 13:N-(4-(2-((8-hydroxyquinolin-5-yl)methylene)hydrazine-1-carbonyl)phenyl)thiophene-2-sulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.71 (s, 1H), 10.90 (s, 1H), 10.44 (br s,1H), 9.58 (d, J=8.7 Hz, 1H), 8.93 (d, J=2.7 Hz, 1H), 8.77 (s, 1H), 7.94(dd, J=5.0, 1.3 Hz, 1H), 7.81-7.91 (m, 2H), 7.69-7.81 (m, 2H), 7.67 (dd,J=3.7, 1.3 Hz, 1H), 7.23-7.35 (m, 2H), 7.08-7.20 (m, 2H); MS (ESI)C₂₁H₁₅N₄O₄S₂[M-H]⁻ m/z expected=451.1, observed=451.0; HPLC-1=99%;HPLC-2=99%.

Example 15

Analog 14:4-bromo-N-(4-(2-((8-hydroxyquinolin-5-yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.71 (s, 1H), 10.86 (s, 1H), 10.44 (br s,1H), 9.57 (d, J=8.6 Hz, 1H), 8.93 (d, J=2.7 Hz, 1H), 8.78 (s, 1H),7.66-7.91 (m, 8H), 7.23 (d, J=8.6 Hz, 2H), 7.09-7.18 (m, 1H), 8.96 (s,1H); MS (ESI) C₂₃H₁₆BrN₄O₄S [M-H]⁻ m/z expected=523.0, observed=522.9;HPLC-1=99%; HPLC-2=99%.

Example 16

Analog 15:4-ethoxy-N-(4-(2-((8-hydroxyquinolin-5-yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.67 (s, 1H), 10.63 (s, 1H), 10.43 (br s,1H), 9.57 (d, J=7.8 Hz, 1H), 8.93 (d, J=2.8 Hz, 1H), 8.76 (s, 1H),7.67-7.86 (m, 6H), 7.22 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.0 Hz, 1H), 7.06(d, J=8.9 Hz, 2H), 4.06 (q, J=6.9 Hz, 2H), 1.30 (t, J=6.9 Hz, 3H); MS(ESI) C₂₅H₂₁N₄O₅S [M-H]⁻ m/z expected=489.1, observed=489.0; HPLC-1=98%;HPLC-2=98%.

Example 17

Analog 16: N′-((5-nitrofuran-2-yl)methylene)thiophene-2-carbohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.26 (s, 1H), 8.35 (br s, 1H), 7.96 (br s,2H), 7.81 (d, J=4.0 Hz, 1H), 7.28 (d, J=4.0 Hz, 1H), 7.24 (dd, J=4.9,3.9 Hz, 1H); MS (ESI) C₁₀H₈N₃O₃S [MH]⁺ m/z expected=266.0,observed=266.1; HPLC-1=98%; HPLC-2=98%.

Example 18

Analog 17:5-chloro-N′-((5-nitrofuran-2-yl)methylene)thiophene-2-carbohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.38 (s, 1H), 8.34 (br s, 0.3H), 8.03 (brs, 0.7H), 7.89 (br s, 0.7H), 7.80 (d, J=3.9 Hz, 1.3H), 7.24-7.33 (m,2.2H)—Putatively mixture of ˜2:1 rotamers; MS (ESI) C₁₀H₇N₃O₄S [MH]⁺ m/zexpected=300.0, observed=300.0; HPLC-1=98%; HPLC-2=98%.

Example 19

Analog 18: N′-((5-nitrofuran-2-yl)methylene)benzohydrazide. ¹H-NMR (300MHz, d₆-DMSO) δ 12.25 (s, 1H), 8.40 (br s, 1H), 7.91 (d, J=7.2 Hz, 2H),7.80 (d, J=3.9 Hz, 1H), 7.59-7.67 (m, 1H), 7.50-7.59 (m, 2H), 7.28 (d,J=3.6 Hz, 1H); MS (ESI) C₁₂H₁₀N₃O₄[MH]⁺ m/z expected=260.0,observed=260.1; HPLC-1=>99%; HPLC-2=98%.

Example 20

Analog 19: N′-((5-nitrofuran-2-yl)methylene)isonicotinohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 12.44 (s, 1H), 8.81 (d, J=5.6 Hz, 2H), 8.41 (s,1H), 7.75-7.89 (m, 3H), 7.33 (d, J=3.8 Hz, 1H); MS (ESI) C₁₁H₉N₄O₄ [MH]⁺m/z expected=261.1, observed=261.1; HPLC-1=99%; HPLC-2=99%.

Example 21

Analog 20: 4-methoxy-N′-((5-nitrofuran-2-yl)methylene)benzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.12 (s, 1H), 8.39 (br s, 1H), 7.88-7.96(m, 2H), 7.80 (d, J=4.0 Hz, 1H), 7.26 (d, J=3.9 Hz, 1H), 7.05-7.12 (m,2H), 3.84 (s, 3H); MS (ESI) C₁₃H₁₀N₃O₅ [M-H]⁻ m/z expected=288.1,observed=288.0; HPLC-1=>99%; HPLC-2=>99%.

Example 22

Analog 21:4-(dimethylamino)-N′-((5-nitrofuran-2-yl)methylene)benzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.95 (s, 1H), 8.38 (s, 1H), 7.77-7.85 (m,3H), 7.22 (d, J=3.9 Hz, 1H), 6.73-6.81 (m, 2H), 3.01 (s, 6H); MS (ESI)C₁₄H₁₅N₄O₄ [MH]⁺ m/z expected=303.1, observed=303.1; HPLC-1=>99%;HPLC-2=99%.

Example 23

Analog 22: N′-((5-nitrofuran-2-yl)methylene)-1-naphthohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 12.26 (s, 1H), 8.30 (s, 1H), 8.18-8.25 (m, 1H),8.13 (d, J=8.2 Hz, 1H), 7.99-8.08 (m, 1H), 7.76-7.86 (m, 2H), 7.57-7.67(m, 3H), 7.30 (d, J=3.9 Hz, 1H); MS (ESI) C₁₆H₁₂N₃O₄ [M-H]⁻ m/zexpected=310.1, observed=310.0; HPLC-1=99%; HPLC-2=98%.

Example 24

Analog 23: N′-((5-nitrofuran-2-yl)methylene)-2-phenoxybenzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.17 (s, 1H), 8.26 (s, 0.7H), 7.92 (s,0.3H), 7.78 (d, J=3.8 Hz, 0.7H), 7.70-7.75 (m, 0.4H), 7.67 (d, J=7.3 Hz,0.7H), 7.34-7.58 (m, 2.9H), 6.94-7.33 (m, 7.0H), 6.893-6.91 (m,0.4H)—Putatively mixture of ˜2:1 rotamers; MS (ESI) C₁₈H₁₄N₃O₅ [MH]⁺ m/zexpected=352.1, observed=352.1; HPLC-1=>99%; HPLC-2=98%.

Example 25

Analog 24: N′-((5-nitrofuran-2-yl)methylene)-3-phenoxybenzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.25 (s, 1H), 8.39 (s, 1H), 7.80 (d, J=3.9Hz, 1H), 7.70 (d, J=7.6 Hz, 1H), 7.51-7.61 (m, 2H), 7.38-7.48 (m, 2H),7.23-7.32 (m, 2H), 7.15-7.23 (m, 1H), 7.04-7.12 (m, 2H); MS (ESI)C₁₈H₁₄N₃O₅ [MH]⁺ m/z expected=352.1, observed=352.1; HPLC-1=>99%;HPLC-2=98%.

Example 26

Analog 25:N,N-diethyl-3-(2-((5-nitrofuran-2-yl)methylene)hydrazine-1-carbonyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.45 (s, 1H), 8.43 (s, 1H), 8.31 (s, 1H),8.20 (d, J=7.2 Hz, 1H), 8.03 (d, J=7.7 Hz, 1H), 7.74-7.85 (m, 2H), 7.31(d, J=3.4 Hz, 1H), 3.20 (q, J=7.1 Hz, 4H), 1.05 (t, J=7.1 Hz, 6H); MS(ESI) C₁₆H₁₉N₄O₆S [MH]⁺ m/z expected=395.1, observed=395.1; HPLC-1=>99%;HPLC-2=>99%.

Example 27

Analog 26:N-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazine-1-carbonyl)phenyl)thiophene-2-sulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.06 (s, 1H), 10.87 (s, 1H), 8.27 (s, 1H),7.85 (dd, J=5.0, 1.4 Hz, 1H), 7.69-7.79 (m, 3H), 7.58 (dd, J=3.8, 1.4Hz, 1H), 7.15-7.25 (m, 3H), 7.06 (dd, J=4.9, 3.8 Hz, 1H); MS (ESI)C₁₆H₁₃N₄O₆S₂[MH]⁺ m/z expected=421.0, observed=421.1; HPLC-1=98%;HPLC-2=99%.

Example 28

Analog 27:4-bromo-N-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.11 (s, 1H), 10.89 (s, 1H), 8.34 (s, 1H),7.70-7.87 (m, 7H), 7.18-7.31 (m, 3H); MS (ESI) C₁₈H₁₄BrN₄O₆S [MH]⁺ m/zexpected=495.0, observed=494.9; HPLC-1=98%; HPLC-2=97%.

Example 29

Analog 28:4-ethoxy-N-(4-(2-((5-nitrofuran-2-yl)methylene)hydrazine-1-carbonyl)phenyl)benzenesulfonamide.¹H-NMR (300 MHz, d₆-DMSO) δ 12.09 (s, 1H), 10.68 (s, 1H), 8.34 (s, 1H),7.68-7.86 (m, 5H), 7.17-7.29 (m, 3H), 7.01-7.10 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 1.30 (t, J=1.3 Hz, 3H); MS (ESI) C₂₀H₁₉N₄O₇S [MH]⁺ m/zexpected=459.1, observed=459.0; HPLC-1=97%; HPLC-2=97%.

Example 30

Analog 29:N′-((4-hydroxynaphthalen-1-yl)methylene)-4-methoxybenzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.57 (s, 1H), 10.75 (s, 1H), 8.82-8.99 (m, 2H),8.20 (d, J=8.1 Hz, 1H), 7.84-7.99 (m, 2H), 7.69 (d, J=7.8 Hz, 1H), 7.61(t, J=7.5 Hz, 1H), 7.50 (t, J=7.5 Hz, 1H), 6.99-7.11 (m, 2H), 6.93 (d,J=7.9 Hz, 1H), 3.81 (s, 3H); MS (ESI) C₁₉H₁₇N₂O₃ [M-H]⁻ m/zexpected=321.1, observed=321.1; HPLC-1=99%; HPLC-2=99%.

Example 31

Analog 30: N′-((4-hydroxynaphthalen-1-yl)methylene)benzohydrazide.¹H-NMR (300 MHz, d₆-DMSO) δ 11.72 (s, 1H), 10.81 (s, 1H), 8.89-9.02 (m,2H), 8.24 (d, J=8.1 Hz, 1H), 7.95 (d, J=7.2 Hz, 2H), 7.74 (d, J=8.1 Hz,1H), 7.48-7.70 (m, 5H), 6.97 (d, J=7.9 Hz, 1H); MS (ESI) C₁₈H₁₅N₂O₂[MH]⁺ m/z expected=291.1, observed=291.1; HPLC-1=99%; HPLC-2=98%.

Example 32

Analog 31: 4-methoxy-N′-(quinolin-5-ylmethylene)benzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.91 (s, 1H), 9.45 (d, J=8.3 Hz, 1H), 8.98 (d,J=3.9 Hz, 2H), 8.10 (d, J=8.1 Hz, 1H), 7.91-8.02 (m, 3H), 7.80-7.89 (m,1H), 7.68 (dd, J=8.1, 3.7 Hz, 1H), 7.10 (d, J=8.8 Hz, 2H), 3.85 (s, 3H);MS (ESI) C₁₈H₁₆N₃O₂ [MH]⁺ m/z expected=306.1, observed=306.1;HPLC-1=99%; HPLC-2=99%.

Example 33

Analog 32: N′-(quinolin-5-ylmethylene)benzohydrazide. ¹H-NMR (300 MHz,d₆-DMSO) δ 12.05 (s, 1H), 9.45 (d, J=8.5 Hz, 1H), 8.93-9.06 (m, 2H),8.10 (d, J=8.2 Hz, 1H), 7.97 (t, J=7.5 Hz, 3H), 7.80-7.89 (m, 1H), 7.69(dd, J=8.7, 4.0 Hz, 1H), 7.50-7.64 (m, 3H); MS (ESI) C₁₇H₁₄N₃O [MH]⁺ m/zexpected=276.1, observed=276.1; HPLC-1=>99%; HPLC-2=>99%.

Example 34

Analog 33: 4-methoxy-N′-(naphthalen-1-ylmethylene)benzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.83 (s, 1H), 9.10 (s, 1H), 8.87 (d, J=8.2 Hz,1H), 8.02 (d, J=7.9 Hz, 2H), 7.89-7.99 (m, 3H), 7.57-7.72 (m, 3H),7.06-7.13 (m, 2H), 3.85 (s, 3H); MS (ESI) C₁₉H₁₇N₂O₂ [MH]⁺ m/zexpected=305.1, observed=305.1; HPLC-1=f>99%; HPLC-2=>99%.

Example 35

Analog 34: N′-(naphthalen-1-ylmethylene)benzohydrazide. ¹H-NMR (300 MHz,d₆-DMSO) δ 11.96 (s, 1H), 9.12 (s, 1H), 8.88 (d, J=8.3 Hz, 1H), 8.03(dd, J=7.7, 3.2 Hz, 2H), 7.91-8.00 (m, 3H), 7.52-7.73 (m, 6H); MS (ESI)C₁₈H₁₅N₂O [MH]⁺ m/z expected=275.1, observed=275.0; HPLC-1=>99%;HPLC-2=>99%.

Example 36

Analog 35: N′-(4-hydroxybenzylidene)-4-methoxybenzohydrazide. ¹H-NMR(300 MHz, d₆-DMSO) δ 11.53 (s, 1H), 9.93 (s, 1H), 8.33 (s, 1H), 7.89 (d,J=8.8 Hz, 2H), 7.50-7.59 (m, 2H), 7.00-7.09 (m, 2H), 6.83 (d, J=8.5 Hz,2H), 3.83 (s, 3H); MS (ESI) C₁₅H₁₅N₂O₃ [MH]⁺ m/z expected=271.1,observed=271.1; HPLC-1=95%; HPLC-2=99%.

Example 37

Analog 36: N′-(4-hydroxybenzylidene)benzohydrazide. ¹H-NMR (300 MHz,d₆-DMSO) δ 11.65 (s, 1H), 9.95 (s, 1H), 8.34 (s, 1H), 7.89 (d, J=6.8 Hz,2H), 7.44-7.64 (m, 5H), 6.84 (d, J=8.6 Hz, 2H); MS (ESI) C₁₄H₁₃N₂O₂[MH]⁺ m/z expected=241.1, observed=241.1; HPLC-1=95%; HPLC-2=98%.

Example 38

Analog 37: N′-benzylidene-4-methoxybenzohydrazide. ¹H-NMR (300 MHz,d₆-DMSO) δ 11.74 (s, 1H), 8.45 (s, 1H), 7.87-7.96 (m, 2H), 7.67-7.77 (m,2H), 7.39-7.51 (m, 3H), 7.02-7.11 (m, 2H), 3.83 (s, 3H); MS (ESI)C₁₅H₁₅N₂O₂ [MH]⁺ m/z expected=255.1, observed=255.1; HPLC-1=99%;HPLC-2=99%.

Example 39

Analog 38: N′-benzylidenebenzohydrazide. ¹H-NMR (300 MHz, d₆-DMSO) δ11.87 (s, 1H), 8.47 (s, 1H), 7.92 (d, J=7.0 Hz, 2H), 7.69-7.78 (m, 2H),7.41-7.64 (m, 6H); MS (ESI) C₁₄H₁₃N₂O [MH]⁺ m/z expected=225.1,observed=225.1; HPLC-1=>99%; HPLC-2=>99%.

Example 40

Analog 39: N′-(furan-2-ylmethylene)-4-methoxybenzohydrazide. ¹H-NMR (300MHz, d₆-DMSO) δ 11.68 (s, 1H), 8.33 (s, 1H), 7.86-7.92 (m, 2H), 7.84 (s,1H), 7.02-7.10 (m, 2H), 6.91 (d, J=3.1 Hz, 1H), 6.63 (dd, J=3.3, 1.7 Hz,1H), 3.83 (s, 3H); MS (ESI) C₁₃H₁₃N₂O₃ [MH]⁺ m/z expected=245.1,observed=245.1; HPLC-1=98%; HPLC-2=98%.

Example 41

General materials and methods for biochemical & cell-based experiments.

The bacterial proliferation assays employed the following bacterialstrains: NEB 5-alpha Escherichia coli (a derivative of DH5α E. coli, NewEngland Biolabs #C2987H); Enterococcus faecium—(Orla-Jensen) Schleiferand Kilpper-Balz strain NCTC 7171 (ATCC #19434); Staphylococcusaureus—Rosenbranch strain Seattle 1945 (ATCC #25923); Klebsiellapneumoniae—(Schroeter) Trevisan strain NCTC 9633 (ATCC #13883);Acinetobacter baumannii—Bouvet and Grimont strain 2208 (ATCC 19606);Pseudomonas aeruginosa—(Schroeter) Migula strain NCTC 10332 (ATCC#10145); Enterobacter cloacae—E. cloacae, subsp. cloacae (Jordan)Hormaeche and Edwards strain CDC 442-68 (ATCC #13047). For proteinexpression and purification, NEB 5-alpha and BL21 (DE3) E. coli cellswere purchased from New England Biolabs, and Rosetta™ 2 (DE3) E. colicells were purchased from EMD Millipore. The human cell viability assaysused FHC colon cells (CRL-1831) and FHs 74 Int small intestine cells(CCL-241) obtained from the ATCC. Ampicillin was used at a concentrationof 50 μg/mL, when appropriate.

Example 42

Expression and purification of E. coli GroEL and GroES proteins.Referring to FIG. 10, an illustration of the function of the GroEL/EScomplex is provided. E. coli GroEL and GroES were expressed andpurified. E. coli GroEL was expressed from a trc-promoted and Amp(+)resistance marker plasmid in NEB 5-alpha E. coli cells. GroES wasexpressed from a T7-promoted and Amp(+) resistance plasmid in E. coliBL21 (DE3) cells. Transformed colonies were plated ontoAmpicillin-treated LB agar and incubated overnight at 37° C. A singlecolony was selected and grown in LB media treated with Ampicillin for 16hours at 37° C. at 200 rpm. Cells were then sub-cultured in scaled-upAmpicillin-treated LB medium and grown at 37° C. at 230 rpm until anOD₆₀₀ of 0.5 was reached, then were induced with 0.8 mM IPTG andcontinued to grow for ˜2.5 h at 37° C. The cultures were centrifuged at8,000 rpm at 4° C. and the cell pellets were collected and re-suspendedin Buffer A (50 mM Tris-HCl, pH 7.4, and 20 mM NaCl) supplemented withEDTA-free complete protease inhibitor cocktail (Roche). The combinedsuspension was lysed by sonication, the lysate was centrifuged at 14,000rpm at 4° C., and the clarified lysate was passed through a 0.45 μmfilter (Millipore).

Example 43

Anion exchange purification of GroEL: The filtered lysate was loadedonto a GE HiScale Anion exchange column (Q Sepharose fast flow anionexchange resin) that was equilibrated with 2 column volumes of Buffer A.The loaded column was washed with 3 column volumes of Buffer Acontaining 28% of Buffer B (50 mM Tris-HCl, pH 7.4, and 1 M NaCl), thenbound protein was eluted with a 28-60% gradient elution of Buffer B over3 column volumes. Protein-containing fractions, as identified bySDS-PAGE, were collected, spin concentrated using a 10 kDa AmiconUltra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4° C. in 50 mMTris-HCl, pH 7.4, and 150 mM NaCl solution.

Example 44

Size exclusion chromatography of GroEL: The dialyzed protein was loadedonto a Superdex 200 size exclusion column (HiLoad 26/600, GE) that wasequilibrated with 2 column volumes of 50 mM Tris-HCl, pH 7.4, and 150 mMNaCl solution. The loaded column was eluted with 1 column volume of 50mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. Protein-containingfractions, as identified by SDS-PAGE, were collected, spin concentratedusing a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), anddialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (ThermoScientific) at 4° C. in 50 mM Tris-HCl, pH 7.4, and 150 mM NaClsolution. The final protein concentration was determined by CoomassieProtein Assay Kit (Thermo Scientific). Batches of GroEL protein fortesting were stored at 4° C. for up to one month, then discarded.

Example 45

Anion exchange purification of GroES: The filtered lysate was loadedonto a GE HiScale anion exchange column (Q Sepharose fast flow anionexchange resin) that was equilibrated with 2 column volumes of Buffer A.The loaded column was washed with 3 column volumes of Buffer Acontaining 10% of Buffer B (50 mM Tris-HCl, pH 7.4, and 1 M NaCl), thenbound protein was eluted with a 10-50% gradient elution of Buffer B over3 column volumes. Protein-containing fractions, as identified bySDS-PAGE, were collected, spin concentrated using a 10 kDa AmiconUltra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4° C. in 50 mMTris-HCl, pH 7.4, and 150 mM NaCl solution.

Example 46

Size exclusion chromatography of GroES: The dialyzed protein was loadedonto a Superdex 200 size exclusion column (HiLoad 26/600, GE) that wasequilibrated with 2 column volumes of 50 mM Tris-HCl, pH 7.4, and 150 mMNaCl solution. The loaded column was eluted with 1 column volume of 50mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. Protein-containingfractions, as identified by SDS-PAGE, were collected, spin concentratedusing a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), anddialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (ThermoScientific) at 4° C. in 50 mM Tris-HCl, pH 7.4, and 150 mM NaClsolution. The final protein concentration was determined by CoomassieProtein Assay Kit (Thermo Scientific). Batches of GroES protein fortesting were stored at 4° C. for up to one month, then discarded.

Example 47

Evaluating compounds for inhibition in the GroEL/ES-mediated dMDHrefolding assay. All compounds were evaluated for inhibiting E. coliGroEL/ES-mediated refolding of the denatured MDH reporter enzyme.Reagent preparation: For these assays, four primary reagent stocks wereprepared: 1) GroEL/ES-dMDH binary complex stock; 2) ATP initiationstock; 3) EDTA quench stock; 4) MDH enzymatic assay stock. Denatured MDH(dMDH) was prepared by 2-fold dilution of MDH (5 mg/ml, soluble pigheart MDH from Roche, product #10127248001) with denaturant buffer (7 Mguanidine-HCl, 200 mM Tris, pH 7.4, and 50 mM DTT). MDH was completelydenatured by incubating at room temperature for >45 min. The binarycomplex solutions were prepared by slowly adding the dMDH stock to astirring stock with GroEL and GroES in folding buffer (50 mM Tris-HCl,pH 7.4, 50 mM KCl, 10 mM MgC₂, and 1 mM DTT). The binary complex stockswere prepared immediately prior to dispensing into the assay plates andhad final protein concentrations of 83.3 nM GroEL, 100 nM GroES, and 20nM dMDH in folding buffer. For the ATP initiation stock, ATP solid wasdiluted into folding buffer to a final concentration of 2.5 mM. Quenchsolution contained 600 mM EDTA (pH 8.0). The MDH enzymatic assay stockconsisted of 20 mM sodium mesoxalate and 1.6 mM NADH in reaction buffer(50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT).

Example 48

Assay Protocol: First, 30 μL aliquots of the GroEL/ES-dMDH binarycomplex stocks were dispensed into clear, 384-well polystyrene plates.Next, 0.5 μL of the compound stocks (10 mM to 4.6 μM, 3-fold dilutionsseries in DMSO) were added by pin-transfer (V&P Scientific). Thechaperonin-mediated refolding cycles were initiated by addition of 20 μLof ATP stock (reagent concentrations during refolding cycle: 50 nMGroEL, 60 nM GroES, 12 nM dMDH, 1 mM ATP, and compounds of 100 μM to 46nM, 3-fold dilution series) and the refolding reactions were incubatedat 37° C. The incubation time was determined from refolding time-coursecontrol experiments until they reached ˜90% completion of the refoldingof dMDH—generally ˜20-40 min. Next, the assay was quenched by additionof 10 μL of the EDTA stock to give a final concentration of 100 mM inthe wells. Enzymatic activity of the refolded MDH was initiated byaddition of 20 μL MDH enzymatic assay stock, and followed by measuringthe NADH absorbance in each well at 340 nm using a Molecular DevicesSpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD+does not). A_(340 nm) measurements were recorded at 0.5 minutes (startpoint) and at successive time points until the amount of NADH consumedreached ˜90% (end point, generally between 20-35 minutes). The ratiobetween start and end point A₃₄₀ values were used to calculate the %inhibition of the GroEL/ES machinery by the compounds IC₅₀ values forthe test compounds were obtained by plotting the % inhibition results inGraphPad Prism and analyzing by non-linear regression using thelog(inhibitor) vs. response (variable slope) equation. Results presentedrepresent the averages of IC₅₀ values obtained from at least fourreplicates in the GroEL/ES-dMDH refolding assay.

Example 49

Counter-screening compounds for inhibition of native MDH enzymaticactivity.

Reagent Preparations & Assay Protocol: This assay was performed asdescribed above for the GroEL/ES-dMDH refolding assay; however, theassay protocol differed in the sequence of compound addition to theassay plates. The refolding reactions were allowed to proceed for 45 minat 37° C. in the absence of test compounds (complete refolding of MDHoccurs), then quenched with the EDTA stock. Compounds were thenpin-transferred into the plates after the EDTA quenching step; thus,compound effects are only possible by inhibiting the fully-refolded MDHreporter substrate. Next, enzymatic activity of the refolded MDH wasinitiated by addition of 20 μL MDH enzymatic assay stock and followed bymeasuring the NADH absorbance in each well at 340 nm using a MolecularDevices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm,while NAD+ does not). A_(340 nm) measurements were recorded at 0.5minutes (start point) and at successive time points until the amount ofNADH consumed reached ˜90% (end point, generally between 20-35 minutes).Compounds were tested in 8-point, 3-fold dilution series (62.5 μM to 29nM during the reporter reaction) in clear, flat-bottom 384-wellmicrotiter plates. IC₅₀ values for the MDH reporter enzyme weredetermined as described above. Results presented represent the averagesof IC₅₀ values obtained from at least five replicates.

Example 50

Evaluating compounds for inhibition in the GroEL/ES-mediated dRhorefolding assay. Reagent preparation: For this assay, five primaryreagent stocks were prepared: 1) GroEL/ES-dRho binary complex stock; 2)ATP initiation stock; 3) thiocyanate enzymatic assay stock; 4)formaldehyde quench stock; 5) ferric nitrate reporter stock. Denaturedrhodanese (dRho) was prepared by 3-fold dilution of rhodanese (Rocheproduct #R¹⁷⁵⁶, diluted to 10 mg/mL with H₂O) with denaturant buffer (12M Urea, 50 mM Tris-HCl, pH 7.4, and 10 mM DTT). Rhodanese was completelydenatured by incubating at room temperature for >45 min. The binarycomplex solution was prepared by slowly adding the dRho stock to astirring stock of concentrated GroEL in modified folding buffer (50 mMTris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl₂, 5 mM Na₂S₂O₃, and 1 mM DTT).The solution was centrifuged at 16,000×g for 5 minutes, and thesupernatant was collected and added to a solution of GroES in modifiedfolding buffer to give final protein concentrations of 100 nM GroEL, 120nM GroES, and 80 nM dRho. The binary complex stock was preparedimmediately prior to use. For the ATP initiation stock, ATP solid wasdiluted into modified folding buffer to a final concentration of 2.0 mM.The thiocyanate enzymatic assay stock was prepared to contain 70 mMKH₂PO₄, 80 mM KCN, and 80 mM Na₂S₂O₃ in water. The formaldehyde quenchsolution contained 30% formaldehyde in water. The ferric nitratereporter stock contained 8.5% w/v Fe(NO₃)₃ and 11.3% v/v HNO₃ in water.

Example 51

Assay Protocol: First, 10 μL aliquots of the GroEL/ES-dRho complex stockwere dispensed into clear, flat-bottom 384-well polystyrene plates.Next, 0.5 μL of the compound stocks (10 mM to 4.6 μM, 3-fold dilutionsin DMSO) were added by pin-transfer. The chaperonin-mediated refoldingcycle was initiated by addition of 10 μL of ATP stock (reagentconcentrations during refolding cycle: 50 nM GroEL, 60 nM GroES, 40 nMdRho, 1 mM ATP, and compounds of 250 μM to 114 nM, 3-fold dilutionseries). After incubating for 40 minutes at 37° C. for the refoldingcycle, 30 μL of the thiocyanate enzymatic assay stock was added andincubated for 60 min at room temperature for the refolded rhodaneseenzymatic reporter reaction. The reporter reaction was quenched byadding 10 μL of the formaldehyde quench stock, and then 40 μL of theferric nitrate reporter stock was added to quantify the amount ofthiocyanate produced during the enzymatic reporter reaction, which isproportional to the amount of dRho refolded by GroEL/ES. Afterincubating at room temperature for 15 min, the absorbance by Fe(SCN)₃was measured at 460 nm using a Molecular Devices SpectraMax Plus384microplate reader. A second set of baseline control plates were preparedanalogously, but without GroEL/ES-dRho protein binary solution, tocorrect for possible interference from compound absorbance or turbidity.IC₅₀ values for the test compounds were obtained by plotting the A₄₆₀results in GraphPad Prism and analyzing by non-linear regression usingthe log(inhibitor) vs. response (variable slope) equation. Resultspresented represent the averages of IC₅₀ values obtained from at leastfour replicates.

Example 52

Counter-screening compounds for inhibition of native rhodanese enzymaticactivity. Reagent Preparations & Assay Protocol: Reagents were identicalto those used in the GroEL/ES-dRho refolding assay described above;however, the assay protocol differed in the sequence of compoundaddition to the wells. Compounds were pin-transferred after 50 minuteincubation for the refolding cycle, but prior to the addition of thethiocyanate enzymatic assay stock. Thus, the refolding reactions wereallowed to proceed for 50 min at 37° C. in the absence of testcompounds, but the enzymatic activity of the refolded rhodanese reporterenzyme was monitored in the presence of test compounds (inhibitorconcentration range during the enzymatic reporter reaction is 100 μM to46 nM—3-fold dilutions). IC₅₀ values for the rhodanese reporter enzymewere determined as described above. Results presented represent theaverages of IC₅₀ values obtained from at least four replicates.

Example 53

Evaluating compounds for inhibition in the GroEL/ES-mediated dMDHrefolding assay and native MDH activity counter-screen in the presenceof E. coli NfsB nitroreductase. These assays were performed nearlyidentically to those mentioned above, but with a couple distinctmodifications. For both assays, 10 μg/mL of the E. coli NfsBnitroreductase enzyme (Sigma product #N9284) was added to the initialGroEL/ES-dMDH solution. For the GroEL/ES-dMDH refolding assay, afterstamping with compounds, 10 μL of a 2.4 mM NADH solution was added,followed by a 10 minute incubation period at 37° C. to bioactivate thenitrofuran analogs. The remainder of the assay was conducted as in ourstandard protocol where no NfsB nitroreductase was present. With theextra 10 μL volume from the NADH addition, this made the compoundconcentrations during the refolding cycle part of the assay range from83 μM to 38 nM (3-fold dilution series). For the native MDH activitycounter-screen in the presence of E. coli NfsB nitroreductase, the assaywas performed as described above for the GroEL/ES-dMDH refolding assaywith NfsB; however, compound addition to the assay plates was conductedafter the EDTA quench step, followed by a 10 minute incubation period at37° C. to bioactivate the nitrofuran analogs. With the extra 10 μLvolume from the NADH addition, this made the compound concentrationsduring the enzymatic reporter part of the assay range from 56 μM to 25nM (3-fold dilution series). IC₅₀ values for the test compounds wereobtained by plotting the % inhibition results in GraphPad Prism andanalyzing by non-linear regression using the log(inhibitor) vs. response(variable slope) equation. Results presented represent the averages ofIC₅₀ values obtained from at least four replicates in each assay.

Example 54

Evaluating compounds for inhibition in the GroEL/ES-mediated dRhorefolding assay. All compounds were evaluated for inhibiting E. coliGroEL/ES-mediated refolding of the denatured Rho reporter enzyme as perour previously reported procedure. Results presented represent theaverages of IC₅₀ values obtained from at least four replicates.

Example 55

Counter-screening compounds for inhibition of native rhodanese enzymaticactivity.

All compounds were counter-screened for inhibiting the enzymaticactivity of the native Rho reporter enzyme as per our previouslyreported procedure. Results presented represent the averages of IC₅₀values obtained from at least four replicates.

Example 56

Evaluating compounds for inhibition in the GroEL-mediated ATPase assay.All compounds were evaluated for inhibiting E. coli GroEL-mediatedATPase activity as per our previously reported procedure althoughemploying only GroEL in solution and not containing GroES or denaturedMDH. Evaluating compounds for inhibition in the GroEL-mediated ATPaseassay.

Example 57

Reagent preparation: For this assay, four primary reagent solutions wereprepared: 1) GroEL protein solution; 2) ATP initiation solution; 3) EDTAquench solution; 4) malachite green reporter solution. The GroEL proteinsolution consisted of 100 nM of GroEL in folding buffer (50 mM Tris-HCl,pH 7.4, 50 mM KCl, 10 mM MgCl₂, and 1 mM DTT), which was preparedimmediately prior to dispensing into the assay plates. For the ATPinitiation solution, ATP solid was diluted into folding buffer to afinal concentration of 2 mM. Quench solution contained 300 mM EDTA (pH8.0). The malachite green reporter solution contained 0.034% w/vmalachite green, 1.04% w/v ammonium molybdate, 1% Tween-20, and 1 M HCldissolved in H₂O.

Example 58

Assay Protocol: First, 10 μL aliquots of the GroEL protein solution weredispensed into clear, flat-bottom 384-well polystyrene plates. Next, 0.5μL of the compound stocks (10 mM to 4.6 μM, 3-fold dilutions in DMSO)were added by pin-transfer. GroEL-mediated ATPase activity was initiatedby addition of 10 μL of ATP solution (compound concentrations during theassay ranged from 250 μM to 114 nM). After incubating for 45 minutes at37° C., 10 μL of the EDTA quench solution was added to quench thereaction cycle. Then, 60 μL of the malachite green reporter solution wasadded and the plates were incubated at room temperature for 10 min. Theabsorbance of the malachite green-phosphate complex was measured at 600nm using a Molecular Devices SpectraMax Plus384 microplate reader. Asecond set of baseline control plates were prepared analogously, butwithout GroEL protein solution, to correct for possible interferencefrom compound absorbance or turbidity. IC₅₀ values for the testcompounds were obtained by plotting the A₆₀₀ results in GraphPad Prismand analyzing by non-linear regression using the log(inhibitor) vs.response (variable slope) equation. Results presented represent theaverages of IC₅₀ values obtained from at least four replicates.

Example 59

Evaluating compounds for inhibition of bacterial cell proliferation.

All compounds were evaluated for inhibiting the proliferation of E. coliand each of the ESKAPE bacteria. Results presented represent theaverages of EC₅₀ values obtained from at least four replicates.Evaluating compounds for inhibition of bacterial cell proliferation.Bacterial Strains: NEB 5-alpha Escherichia coli (a derivative of DH5α E.coli, New England Biolabs #C2987H); Enterococcus faecium—(Orla-Jensen)Schleifer and Kilpper-Balz strain NCTC 7171 (ATCC #19434);Staphylococcus aureus—Rosenbranch strain Seattle 1945 (ATCC #25923);Klebsiella pneumoniae—(Schroeter) Trevisan strain NCTC 9633 (ATCC#13883); Acinetobacter baumannii—Bouvet and Grimont strain 2208 (ATCC19606); Pseudomonas aeruginosa—(Schroeter) Migula strain NCTC 10332(ATCC #10145); Enterobacter cloacae—E. cloacae, subsp. cloacae (Jordan)Hormaeche and Edwards strain CDC 442-68 (ATCC #13047).

Growth Media: E. coli were grown with LB medium and all ESKAPE bacteriawere grown in Brain Heart Infusion (BHI) medium (Becton, Dickinson andCompany), with all liquid cultures supplemented with 25 mg/L Ca²⁺ and12.5 mg/L Mg²⁺ to mimic physiological free concentrations of thesecations. A 10 mg/mL Ca²⁺ stock solution was prepared by dissolving 1.84g of CaCl₂.2H₂O in 50 mL of deionized water, and a 10 mg/mL Mg²⁺ stocksolution was prepared by dissolving 4.18 g of MgCl₂.6H₂O in 50 mLdeionized water—filter sterilized using 0.2 μm cellulose-acetatefilters. 2.5 mL of the sterile 10 mg/mL Ca²⁺ stock and 1.25 mL of thesterile 10 mg/mL Mg²⁺ stock solutions were added per 1 L of autoclavedLB or BHI media to obtain 25 mg/L Ca²⁺ and 12.5 mg/L Mg²⁺ ions,respectively.

Example 60

General Assay Protocol: Stock bacterial cultures were streaked onto LBor BHI agar plates and grown overnight at 37° C. Fresh aliquots ofcation supplemented media were inoculated with single bacterial coloniesand the cultures were grown overnight at 37° C. with shaking (240 rpm).The following morning, cultures were diluted 10-fold into fresh mediaand grown at 37° C. until bacteria had reached mid-log phase growth(OD₆₀₀˜0.4-0.6). The cultures were then diluted into fresh media toachieve final OD₆₀₀ readings of 0.017. Aliquots of these dilutedcultures (30 μL) were added to clear, flat-bottom 384-well polystyreneplates that were previously stamped with 0.5 μL of test compounds in 20μL of media (yielding initial OD₆₀₀=0.01 bacterial cultures). Allcompounds were tested in dose-response with concentration ranges duringthe proliferation assays from 100 μM to 46 nM (3-fold dilution series).A second set of baseline control plates were prepared analogously, butwithout any bacteria added, to correct for possible compound absorbanceand/or precipitation. Plates were sealed with “Breathe Easy” oxygenpermeable membranes (Diversified Biotech) and left to incubate at 37° C.without shaking (stagnant assay) until the bacteria had reached mid-logphase growth. Plates were then read at 600 nm using a Molecular DevicesSpectraMax Plus384 microplate reader. EC₅₀ values for the test compoundswere obtained by plotting the OD₆₀₀ results in GraphPad Prism andanalyzing by non-linear regression using the log(inhibitor) vs. response(variable slope) equation. Results presented represent the averages ofEC₅₀ values obtained from at least four replicates.

Examples 61

Evaluating compound effects on the viability of human colon and smallintestine cells. All compounds were evaluated for cytotoxicity to humancolon (FHC) and small intestine (FHs 74 Int) cells using an AlamarBlue-based viability assay. Results presented represent the averages ofCC₅₀ values obtained from at least four replicates for the FHC and threereplicates for the FHs 74 Int cell lines. Evaluating compound effects onthe viability of human colon and small intestine cells. Evaluation ofcompound cytotoxicities to FHC colon and FHs 74 Int small intestinecells were performed using Alamar Blue-based viability assays. FHC cellswere maintained in Hyclone DMEM:F12 1:1 medium containing L-glutamineand supplemented with 10 mM HEPES, 10 ng/mL cholera toxin, 0.005 mg/mLinsulin, 0.005 mg/mL transferrin, 100 ng/mL hydrocortisone, and 10% FBS(Midwest Scientific, USDAFBS). FHs 74 Int cells were maintained inHybri-care medium (ATCC 46-X) supplemented with 30 ng/mL recombinantepidermal growth factor (EGF) and 10% FBS (Midwest Scientific, USDAFBS).All assays were carried out in 384-well plates (BRAND cell culture gradeplates, 781980). Cells at 90% confluence were harvested and diluted ingrowth medium, then 45 μL of the FHC cells (1,500 cells/well) or FHs 74Int cells (1,500 cells/well) were dispensed per well. Plates were sealedwith “Breathe Easy” oxygen permeable membranes (Diversified Biotech) andincubated at 37° C., 5% CO₂, for 24 h. The following day, 1 μL of thecompound stocks (10 mM to 4.6 μM, 3-fold dilutions in DMSO) werepre-diluted by pin-transfer into 25 μL of the respective growth media,then 15 μL aliquots of these solutions were added to the cell assayplates to give inhibitor concentration ranges of 100 μM to 46 nM duringthe assay (final DMSO concentration of 1% was maintained during theassay). Plates were sealed with “Breathe Easy” oxygen permeablemembranes and incubated for an additional 48 h at 37° C. and 5% CO₂. TheAlamar Blue reporter reagents were then added to a final concentrationof 10%, and the plates incubated at 37° C. and 5% CO₂ while the reporterreagents developed. Sample fluorescence (535 nm excitation, 590 nmemission) was read over time using a Molecular Devices FlexStation II384-well plate reader (readings taken between 4-24 h of incubation so asto achieve signals in the 30-60% range for conversion of resazurin toresorufin). Cell viability was calculated as per vendor instructions(Thermo Fisher—Alamar Blue cell viability assay manual). CytotoxicityCC₅₀ values for the test compounds were obtained by plotting the %resazurin reduction results in GraphPad Prism and analyzing bynon-linear regression using the log(inhibitor) vs. response (variableslope) equation. Results presented represent the averages of CC₅₀ valuesobtained from at least four replicates for the FHC and three replicatesfor the FHs 74 Int cell lines.

Example 62

Evaluating the ability of E. coli to generate resistance to leadinhibitors.

To identify potential resistance toward nifuroxazide, nitrofurantoin,and lead inhibitor 17, a liquid culture, 12-day serial passage assay.Evaluating the ability of E. coli to generate resistance to leadinhibitors. To identify potential resistance toward 17, nifuroxazide,and nitrofurantoin, a liquid culture, 12-day serial passage assay wasemployed (each compound was tested in duplicate). NEB 5-alpha E. colibacteria were streaked onto an LB agar plate and grown overnight at 37°C. Two fresh aliquots of LB media (supplemented with 25 mg/L Ca²⁺ and12.5 mg/L Mg²⁺) were inoculated with separate bacterial colonies and thecultures were grown overnight at 37° C., with shaking (240 rpm). Theovernight cultures were then sub-cultured (10× dilution) into freshmedia solutions and grown at 37° C. to mid-log phase, then diluted intofresh media to achieve two separate cultures with final OD₆₀₀ readingsof 0.01. Aliquots of the diluted cultures (200 μL) were dispensed into aclear, 96-well polystyrene plate (one culture was used for replicate 1for each compound, and the other culture was used for replicate 2 foreach compound), followed by the addition of 2 μL of nifuroxazide,nitrofurantoin, and compound 17 in DMSO. Test compounds were evaluatedin duplicate with concentration ranges during the resistance assay from100 μM to 48.8 nM, 2-fold dilution series. Plates were sealed with“Breathe Easy” oxygen permeable membranes (Diversified Biotech) and leftto incubate at 37° C. without shaking (stagnant assay). OD₆₀₀ readingswere taken at the 24 h time point to monitor for bacterial growth. Asecond baseline control plate was prepared analogously, without anybacteria added, to correct for possible compound absorbance and/orprecipitation. For inoculations on subsequent days, bacteria from thewells with the highest test compound concentration where the OD₆₀₀was >0.2, were sub-cultured in 5 mL of fresh media at 37° C. untilmid-log phase growth was reached. Separate cultures were maintained fromthis point forward for each of the compound replicates—i.e. 6 culturescontinually propagated. The bacteria were then diluted into fresh mediato OD₆₀₀ of 0.01 and the bacteria were propagated again as describedabove. This procedure was repeated each day, for a total of 12 days, toobserve changes in inhibitor EC₅₀ values over each passage (aliquots ofeach of these daily cultures were flash frozen in liquid nitrogen andstored at −80° C. for future use). Inhibitor EC₅₀ values were obtainedby plotting the OD₆₀₀ results from each passage in GraphPad Prism andanalyzing by non-linear regression using the log(inhibitor) vs. response(variable slope) equation.

Example 63

Control compounds, calculation of IC₅₀/EC₅₀/CC₅₀ values, and statisticalconsiderations. For all assays, DMSO was used as negative control and apanel of our previously discovered and reported chaperonin inhibitorswere used as positive controls: e.g. compounds 8, 9, and 18 from Johnsonet. al 2014 and Abdeen et. al 2016; suramin and compound 2h-p fromAbdeen et. al 2016; compounds 20R, 20L, and 28R from Abdeen et. al 2018;and closantel and rafoxanide from Kunkle et. al 2018. Bacterialproliferation assays also included antibiotic controls such asvancomycin, daptomycin, and rifampicin. All IC₅₀/EC₅₀/CC₅₀ resultsreported are averages of values determined from individual dose-responsecurves in assay replicates as follows: 1) individual I/E/CC₅₀ valuesfrom assay replicates were first log-transformed and the averagelog(I/E/CC₅₀) values and standard deviations (SD) calculated; 2)replicate log(I/E/CC₅₀) values were evaluated for outliers using theROUT method in GraphPad Prism (Q of 10%); and 3) average I/E/CC₅₀ valueswere then back-calculated from the average log(I/E/CC₅₀) values. Tocompare log(IC₅₀) values between different assays, two-tailed Spearmancorrelation analyses were performed using GraphPad Prism (95% confidencelevel). For compounds where log(I/E/CC₅₀) values were greater than themaximum compound concentrations tested (i.e. >1.75, >1.80, >1.92, >2.00,and >2.40—or >56, >63, >83, >100, and >250 μM, respectively), resultswere represented as 0.1 log units higher than the maximum concentrationstested (i.e. 1.85, 1.90, 2.02, 2.10, and 2.50—or 71, 79, 105, 126, and316 μM, respectively) so as not to overly bias results because of theunavailability of definitive values for inactive compounds.

Example 64

Previous studies identified bis-sulfonamido-2-phenylbenzoxazole (BSP)and salicylanilides (SCA) analogs that were potent GroEL/ES inhibitorswith antibacterial activity against Gram-positive bacteria. Compound 1was previously found to be a moderate inhibitor of GroEL/ES-mediatedrefolding of denatured rhodanese and malate dehydrogenase substrates(IC₅₀=18 & 31 μM, respectively) and a weak to moderate inhibitor of theproliferation of B. subtilis (EC₅₀=83 μM), methicillin-resistant S.aureus (EC₅₀=56 μM), K. pneumoniae (EC₅₀=95 μM), A. baumannii (EC₅₀=32μM), and SM101 E. coli (EC₅₀=19 μM). Owing to its ability to inhibitboth Gram-positive and Gram-negative bacteria, and its structuralsimilarity to known antibacterials nitroxoline, nifuroxazide, andnitrofurantoin, in this study, we sought to develop new compound 1analogs (Scheme 1) that were more potent and selective inhibitors ofGroEL/ES and bacterial proliferation.

Based on the similarities of compound 1 with nitroxoline, nifuroxazide,and nitrofurantoin, we developed a library of analogs that probed thestructure-activity relationships (SAR) of the cyclic/aryl substructureson both the right and left-hand sides of the N-acylhydrazone linker(Scheme 1). The right-hand substructure of these analogs containedeither a hydroxyquinoline group (mimicking 1 and nitroxoline) or anitrofuran group (mimicking nifuroxazide and nitrofurantoin), generatingtwo distinct series of compounds. For each series, 15 left-side groupswere assessed that contained a diverse range of substructures, includingsome that we have found effective with other GroEL/ES inhibitorscaffolds that have shown antibacterial properties (e.g. thiophenes,2-chlorothiophenes, and aryl-sulfonamides). These analogs were tested ina panel of in vitro assays for bacterial proliferation inhibition,GroEL/ES inhibition (both substrate folding and ATPase functions), humancell cytotoxicity, and bacterial resistance towards lead analogs. Theresults from testing analogs in this panel of biochemical and cell-basedexperiments are presented herein, with a discussion on SAR and thepotential of these series as GroEL/ES-targeting antibacterialcandidates.

Example 65

Conceptualizing and developing the hydroxyquinoline andnitrofuran-containing series of compound 1 analogs. Owing to theirsimilarity to the known antibacterials nitroxoline, nifuroxazide, andnitrofurantoin, we investigated two primary series of compound 1 analogsthat also bore hydroxyquinoline and nitrofuran substructures (Scheme 1as shown in Example 64). For each series, 15 left-side groups wereassessed that contain a diverse range of substructures, including somethat we have found effective with other GroEL/ES inhibitor scaffoldsthat have shown antibacterial properties (e.g. thiophenes,2-chlorothiophenes, and aryl-sulfonamides). A third group of analogs(29-42) was also investigated to determine which parts of thehydroxyquinoline and nitrofuran aryls were required for inhibitorpotency in the respective assays (refer to Tables 4 and 7 for thestructures of compounds 29-42).

Example 66

Analogs were all synthesized through a one-step coupling reactionbetween the respective aryl-aldehydes and N-acylhydrazides in DMSO, withHCl as a catalyst. After stirring overnight at room temperature, thefinal N-acylhydrazone products were precipitated through the addition ofwater, and the solids were filtered, rinsed, and dried in vacuo. Wherenecessary, compounds were further purified via normal and/orreverse-phase chromatography. Synthesized analogs were analyzed byRP-HPLC for purity and LC-MS and ¹H-NMR for structural. While allcompounds were found to be >95% pure using two distinct sets of RP-HPLCconditions, for some analogs (3, 4, 6, 9, 10, 17, 23), we noticed asplitting of peaks in the ¹H-NMR spectra. This phenomenon has previouslybeen studied by others and reported as resulting from hindered rotationaround the amide bond, providing rotational isomers (rotamers). Thus, webelieve that the purity of these compounds is consistent with HPLCresults showing >95% purity.

Example 67

Evaluating analogs for inhibiting the growth of E. coli and the ESKAPEbacteria. Analogs were initially tested for antibacterial efficacyagainst representative strains of antibiotic-sensitive E. coli and theESKAPE bacteria. To determine compound efficacy, bacterial proliferationassays were carried out in liquid media culture supplemented withphysiological concentrations of free calcium and magnesium cations. Forthese assays, bacterial cultures (OD₆₀₀=0.01) were exposed to testcompounds in 8-point, 3-fold dilution series (100 μM to 46 nMconcentration range), in 384-well plates. After addition of compounds tocultures, the plates were sealed with Breathe-Easy gas permeablemembranes and allowed to grow to mid-log phase (OD₆₀₀˜0.4-0.6),whereupon final OD₆₀₀ readings were taken to assess for bacterial growthand inhibition. Calculated EC₅₀ values are reported in Tables 1, 5, 6,and 7. For easier visualization of results, tables are heat-mappedaccording to inhibitor potencies, with darker cells representing themost potent analogs, and lighter cells the least potent inhibitors.

TABLE 1 Compilation of EC₅₀ values for the hydroxyquinoline andnitrofuran analogs tested in the ESKAPE and E. coli bacterialproliferation assays. Cells that are shaded darker grey are most potent,while those that are shaded lighter grey to white are less potent toinactive (>100 μM). E. S. K. A. P. E. E. # faecium aureus pneumoniaebaumannii aeruginosa cloacae coli Hydroxyquinolines 2 >10037 >100 >100 >100 >100 80 3 >100 63 99 74 >100 92 77 4 7084 >100 >100 >100 >100 >100 5 >100 69 82 56 >100 80 636 >100 >100 >100 >100 >100 >100 79 7 >100 48 87 71 >100 97 46 1 >100 8785 >100 95 100 >100 8 >100 99 >100 >100 >100 >100 93 9 >10056 >100 >100 >100 >100 >100 10 >100 >100 >100 >100 81 >100 >10011 >100 >100 >100 >100 >100 >100 >100 12 >100 >100 >100 >100 85 >100 9913 >100 >100 >100 >100 >100 >100 >100 14 >100 >100 >100 84 >100 >100 10015 >100 >100 >100 >100 >100 >100 >100 Nitroxoline 18 9.5 2.8 2.5 99 6.64.0 Nitrofurans Nitrofurantoin 38 27 40 >100 >100 36 0.69 16 24 7.1 1682 >100 40 0.42 17 12 3.0 19 72 >100 49 0.45 18 31 10 36 >100 >100 >1002.1 19 13 9.5 42 54 >100 73 2.4 Nifuroxazide 8.1 16 37 >100 >100 54 0.8720 22 8.1 >100 >100 >100 >100 1.9 21 >100 34 >100 >100 >100 >100 10.0 2216 6.0 >100 >100 >100 >100 41 23 11 5.2 >100 >100 >100 >100 >100 24 113.5 >100 >100 >100 >100 >100 25 >100 9.6 >100 >100 >100 >100 >100 26 1111 >100 >100 >100 >100 17 27 >100 >100 >100 >100 >100 >100 >10028 >100 >100 >100 >100 >100 >100 >100

Example 68

Results from the bacterial proliferation assays indicated that thehydroxyquinoline analogs were largely ineffective against E. coli andthe ESKAPE pathogens, although several weak inhibitors were identified.Prior to initiating this study, we had held out hope that themetal-chelating properties of the hydroxyquinoline substructure mightallow these inhibitors to act as siderophores that could be activelytaken up into bacteria; however, the lack of efficacy in this series wasperhaps not surprising as a previous study by Pelletier et al. hadindicated that the antibacterial activity of nitroxoline was reducedupon cation supplementation. Despite this setback, we were excited tosee that many of the nitrofuran-based analogs were much more effectiveat inhibiting bacterial growth. In particular, analogs 16-20, 22-24, and26 were moderate to strong inhibitors of the Gram-positive E. faeciumand S. aureus bacteria. For reasons that are not clear, and contrary tothe GroEL/ES inhibition results (discussed below), the dimethylaniline(21) and bulkier sulfonamide-containing analogs (25-28) were lesseffective (or inactive) compared to the analogs with the smallerN-acylhydrazide substructures. Presumably these compounds suffered fromefflux and/or poor permeability through the bacterial cell walls, sincethey were the most potent at inhibiting GroEL/ES and would thus beexpected to be the most potent at killing bacteria if they achievedappreciably high intracellular concentrations, and presuming they wereexhibiting on-target effects against GroEL/ES.

Example 69

While antibacterial effects were limited against the Gram-negative KAPEbacteria, a few analogs (16-21) were potent against E. coli, with EC₅₀values ≤11 μM. Compounds 16 and 17 were more potent than nitroxoline,nifuroxazide, and nitrofurantoin against E. coli (EC₅₀ values <1 μM),and were even moderate inhibitors of K. pneumoniae. These weresignificant findings as our previous studies failed to identify leadanalogs with such high efficacies against E. coli or any of theGram-negative KAPE bacteria. As evidenced by the results of compound 39(Table 7), which has an unsubstituted furan ring, the nitro group wasessential for antibacterial effects. This is putatively becausenitrofuran antibiotics are activated to reactive metabolites bynitroreductases in bacteria, which we discuss further below.Additionally, the hydroxyquinoline and nitrofuran aldehyde startingmaterials (40 and 41, respectively) were potent inhibitors of nearly allthe bacteria (excluding P. aeruginosa), yet the N-acylhydrazone analogswere largely ineffective against the KAPE bacteria, supporting ourbelief that the linkers were not hydrolyzing to their startingmaterials. Therefore, inhibitor potencies likely owed to the finalproducts themselves, or their metabolites in the case of the nitrofuranseries.

Example 70

Evaluating analogs for inhibiting GroEL/ES-mediated substrate refoldingfunctions. Since we identified several analogs that potently inhibitedthe growth of both Gram-positive and Gram-negative bacteria, we nextevaluated their abilities to inhibit E. coli GroEL/ES-mediated substratefolding functions in vitro. We first evaluated all test compounds in ourstandard GroEL/ES-mediated refolding assays that employ either malatedehydrogenase (MDH) or rhodanese (Rho) as the denatured substratereporter enzymes. When denatured, these enzymes are efficiently foldedby GroEL/ES in the absence of inhibitors, and thus act as reporters todetermine the degree of inhibition against the bacterial chaperoninsystem. Inhibition was examined in the presence of these two orthogonalsubstrates in order to support that GroEL/ES inhibitors were on-target.To further support on-target effects against GroEL/ES, wecounter-screened for inhibition of the native MDH and Rho enzymaticreporter reactions, where test compounds were added after the denaturedMDH and Rho substrate enzymes were completely refolded by GroEL/ES. Wehave found that these series of four biochemical assays are highlyeffective at eliminating false-positives as compounds rarely inhibitboth reporter enzymes since their enzymatic read-outs are so differentfrom one another. IC₅₀ results for all compounds tested in these fourassays are presented in Tables 2, 3, and 4 (shown below in Example 77).

Example 71

Unfortunately, we found that the nitrofuran analogs were only weak tomoderate GroEL/ES inhibitors despite being the most potent at inhibitingbacterial growth (see the white diamond symbols in the correlation plotsof FIG. 1A. Conversely, the hydroxyquinoline analogs (black circles inFIG. 1A) were much stronger GroEL/ES inhibitors, although they werelargely inactive against bacteria. While the hydroxyquinolines wereslightly more potent in the GroEL/ES-mediated dRho compared to dMDHrefolding assays (See FIG. 1A), this was likely because several of themhad the coupled effects of also being weak inhibitors of the native Rhoreporter reaction (See FIG. 1B); however, no compounds inhibited nativeMDH, supporting on-target effects against GroEL/ES. Analogs 29-38, wherethe various parts of the hydroxyquinoline substructure were pared away,were largely inactive in all biochemical assays, indicating thenecessity for the complete hydroxyquinoline moiety for inhibition. Asmentioned in the previous section, the dimethylaniline-nitrofuran (21)and bulkier sulfonamide-containing analogs (12-15 and 25-28) weregenerally the most potent GroEL/ES inhibitors, yet the least effectiveor inactive against bacteria, suggesting they were unable to penetratebacteria or were quickly effluxed out—otherwise they would have beenexpected to exhibit stronger antibacterial effects. Although, this ispresuming that the antibacterial effects of these analogs was fromon-target inhibition of GroEL/ES, which remains to be proven.

Example 72

Referring to FIGS. 1A, 1B, and 1C, correlation plots of IC₅₀ values forcompounds evaluated in the respective biochemical assays. FIG. 1A showscompounds inhibited nearly equipotently in the GroEL/ES-dMDH and theGroEL/ES-dRho refolding assays, supporting on-target effects (Spearmancorrelation coefficient comparing log(IC₅₀) values in each assay is0.8877 (p<0.0001)). For the purposes of categorizing inhibitorpotencies, we consider compounds with IC₅₀ values plotted in the greyzones to be inactive (i.e. greater than the maximum concentrationstested), >33 μM to be weak inhibitors, 11-33 μM moderate inhibitors,1-11 μM potent inhibitors, and <1 μM very potent and acting nearstoichiometrically since the concentration of GroEL tetradecamer is 50nM during the refolding cycle (i.e. 700 nM GroEL monomeric subunits).FIG. 1B shows that while some compounds inhibited in the native Rhoenzymatic reporter counter-screen, none inhibited native MDH enzymaticactivity, further supporting on-target effects for inhibiting thechaperonin-mediated refolding cycle. FIG. 1C shows nitrofuran analogsexhibited increased inhibition in the GroEL/ES-dMDH refolding assay inthe presence of E. coli NfsB, while hydroxyquinoline analogs did not(Spearman correlation coefficient comparing hydroxyquinoline log(IC₅₀)values in each assay is 0.9527 (p<0.0001)), supporting a pro-drugmechanism of action through metabolism of the nitro group. No compoundsinhibited native MDH enzymatic activity in either the absence orpresence of E. coli NfsB (See to Tables 2, 3, and 4). Results plotted inthe grey zones represent IC₅₀ values higher than the maximumconcentrations tested. Data points for nifuroxazide (Nfz),nitrofurantoin (Nft), and nitroxoline (Nox) are labelled for comparison.

Example 73

Evaluating analogs for inhibiting GroEL/ES-mediated substrate refoldingfunctions in the presence of the E. coli NfsB type-1 nitroreductase.That none of the nitrofuran analogs were found to be potent GroEL/ESinhibitors is complicated by the fact that nitrofuran-based antibioticsare known to act as prodrugs in vivo—they require metabolism bybacterial nitroreductases to generate reactive metabolites that areassociated with their antibacterial effects. However, our standardGroEL/ES-mediated refolding assays and native substrate reportercounter-screens were conducted without nitroreductases present. Thisemphasized the need to re-examine analogs in modified refolding andnative reporter activity assays that included a nitroreductase enzyme toactivate the nitrofuran analogs in situ, which would be morerepresentative of the bacterial intracellular environment. As an initialtest to see whether or not the activated nitrofuran metabolites would bemore potent GroEL/ES inhibitors, we purchased the E. coli NfsB type 1nitroreductase and modified our standard GroEL/ES-dMDH refolding andnative MDH counter-screens to generate the reactive metabolites in situ(detailed protocols for these assays are presented in the Experimentalsection). We have reported IC₅₀ results from these assays in Tables 2,3, and 4, and graphically in the correlation plot of FIG. 1C, where IC₅₀values are compared between compounds tested in the GroEL/ES-dMDHrefolding assay with and without the E. coli NfsB nitroreductase.

Example 74

In the presence of E. coli NfsB, the nitrofuran analogs exhibiteddramatically increased inhibition of GroEL/ES refolding functions. TheIC₅₀ values for the hydroxyquinoline (1-15) and other analogs withoutnitro groups (e.g. 29-42) were nearly identical in the presence andabsence of NfsB, supporting that increased inhibition was dependent onmodification of the nitrofuran moiety and not other effects on theoverall compound scaffold or from NfsB itself. Furthermore, thenitrofuran metabolites were inactive in the native MDH reportercounter-screen, indicating that increased inhibition was obtainedthrough selectively targeting the GroEL/ES-mediated refolding cycle.While the degree of potency shift varied between analogs, ten shifted toIC₅₀ values ≤11 μM. As points of comparison, the most potent nitrofurananalog in the absence of NfsB had an IC₅₀=26 μM (analog 21), with sixbeing completely inactive (IC₅₀>100 μM). Significant potency shifts wereobserved for the most effective antibacterials (16-20), showing thatsome of the strongest antibacterial compounds were also strong GroEL/ESinhibitors when activated by a nitroreductase enzyme. While activatednifuroxazide and nitrofurantoin were only weak to moderate GroEL/ESinhibitors in this new assay, this may not be surprising as they arereported in the literature to be preferentially activated by the otherE. coli type 1 nitroreductase, NfsA.

Example 75

While we observed a general trend whereby the more potent thatnitrofuran inhibitors were in the in situ NfsB-GroEL/ES-dMDH refoldingassay, the more potent they were at inhibiting S. aureus proliferation(as shown in FIG. 2B), we are guarded as to whether or not inhibitorswere potentially functioning on-target in bacteria. We note thelimitations in the comparison since we had tested such a small set ofanalogs, and only tested them in the presence of E. coli NfsB. As thedifferent nitrofuran analogs would be expected to exhibit varying SARfor activation by NfsA and NfsB, it will be important to test inhibitorsin the presence of both nitroreductases to gain a more complete pictureof how they could be functioning in bacteria. We are in the process ofcloning and expressing E. coli nfsA and nfsB and developing an expandedpanel of nitrofuran-based analogs to study inhibitor mechanisms ingreater detail. Furthermore, investigating the nitroreductases of thevarious ESKAPE bacteria could provide a stronger rationale for why thesecompounds were largely inactive against the KAPE Gram-negative strains.Another limitation was that we were using the E. coli GroEL/ESchaperonin system as a surrogate in our assays. While we anticipateinhibition results will translate to the chaperonin systems of the otherESKAPE bacteria owing to their high sequence similarities (>56% aminoacid identity between the chaperonins from E. coli and the ESKAPEbacteria), this still remains to be demonstrated.

Example 76

Referring to FIGS. 2A, 2B, and 2C, correlation plots comparing IC₅₀values for compounds tested in the in situ NfsB-GroEL/ES-dMDH refoldingassay with EC₅₀ values for inhibiting E. faecium (FIG. 2A), S. aureus(FIG. 2B), and E. coli (FIG. 2C) proliferation. While increasinginhibition by the nitrofurans in the in situ NfsB-GroEL/ES-dMDHrefolding assay in general provided more effective inhibition of S.aureus growth (see FIG. 2B), more thorough studies will need to beconducted—e.g. testing a larger number of analogs in the presence of S.aureus nitroreductases and GroEL/ES chaperonin system—to gain a clearerpicture of whether or not compounds may be functioning on-target againstGroEL/ES in bacteria. Results plotted in the grey zones represent IC₅₀and EC₅₀ values higher than the maximum concentrations tested. Datapoints for nifuroxazide (Nfz), nitrofurantoin (Nft), and nitroxoline(Nox) are labelled for comparison.

Example 77

Evaluating analogs for inhibiting GroEL-mediated ATPase activity. Sincemany proteins use ATP for their biological functions, inhibitingGroEL/ES by competitively binding to the ATP sites could proveproblematic for being able to selectively target the chaperonin system.Thus, we further tested analogs in a well-established GroEL ATPase assaythat employed malachite green to monitor inorganic phosphate liberatedas GroEL hydrolyzed ATP. Briefly, a solution of GroEL was incubated withtest compounds (8-point, 3-fold dilution series) and the assay wasinitiated by addition of ATP. After incubating for 45 minutes, theATPase reaction was quenched by the addition of EDTA. Malachite greenwas then added to the assay to bind and detect free phosphates insolution (absorbance detection at k=600 nm). If analogs inhibited ATPaseactivity, then there would be no free phosphates for malachite green tobind, leading to minimal absorbance at 600 nm. As indicated in Tables 2,3, and 4, none of the analogs from either series inhibited GroEL byblocking ATP hydrolysis. Thus, we believe that these inhibitors bind tosites outside of the ATP pockets. While these results alleviate concernsabout non-selectively targeting other ATP-dependent proteins, we furtherassessed off-target effects through a more definitive approach byevaluating analog cytotoxicity in two human cell lines, discussed below.

TABLE 2 IC₅₀. Biochemical assay IC₅₀ results for hydroxyquinolineanalogs 1-15. Darker grey cell shading indicates compounds with moredesirable bioactivity results (i.e. more potent GroEL/ES inhibitors withlow inhibition of the native MDH and Rho reporter enzymes). BiochemicalAssay IC₅₀ (μM) Native NfsB- Native Native MDH GroEL/ES- GroEL/ES-GroEL/ES- Rho MDH Activity dRho dMDH dMDH GroEL Structure # ActivityActivity w/NfsB Refolding Refolding Refolding ATPase

2 >100 >63 >56 86.4 >100 >83 >250

3 87 >63 >56 38 65 29 >250

4 14 >63 >56 1.4 9.6 4.0 >250

5 95 >63 >56 176 >100 >83 >250

6 >100 >63 >56 >250 >100 >83 >250

7 72 >63 >56 9.0 43 16 >250

1 98 >63 >56 31 77 66 >250

8 67 >63 >56 7.9 27 25 >250

9 97 >63 >56 2.1 13 7.7 >250

10 89 >63 >56 4.5 15 15 >250

11 85 >63 >56 7.8 13 21 >250

12 81 >63 >56 10 19 21 >250

13 >100 >63 >56 0.81 3.2 1.9 >250

14 >100 >63 >56 1.8 14 3.6 >250

15 >100 >63 >56 1.4 6.0 3.1 >250

TABLE 3 IC₅₀. Biochemical assay IC₅₀ results for nitrofuran analogs16-28 plus nitrofurantoin and nifuroxazide. Darker grey cell shadingindicates compounds with more desirable bioactivity results (i.e. morepotent GroEL/ES inhibitors with low inhibition of the native MDH and Rhoreporter enzymes). Biochemical Assay IC₅₀ (μM) Native Native Native MDHRho MDH Activity GroEL/ES-dRho GroEL/ES-dMDH NfsB- GroEL/ES-dMDH GroELStructure # Activity Activity w/NfsB Refolding Refolding RefoldingATPase

Nitrofurantoin >100 >63 >56 >250 >100 84 >250

16 >100 >63 >56 222 >100 7.7 >250

17 56 >63 >56 60 88 3.2 >250

18 84 >63 >56 100 >100 12 >250

19 38 >63 >56 64 >100 23 >250

Nifuroxazide 69 >63 >56 133 >100 19 >250

20 >100 >63 >56 108 69 10 >250

21 >100 >63 >56 26 26 12 >250

22 45 >63 >56 73 82 5.3 >250

23 43 >63 >56 83 85 8.3 >250

24 65 >63 >56 50 71 2.8 >250

25 >100 >63 >56 100 90 6.0 >250

26 44 >63 >56 62 >100 6.0 >250

27 44 >63 >56 59 36 2.0 >250

28 45 >63 >56 66 36 39 >250

TABLE 4 IC₅₀. Biochemical assay IC₅₀ results for additional analogsexamining the substructures of the hydroxyquinoline and nitrofuran arylsthat are important for inhibition. Darker grey cell shading indicatescompounds with more desirable bioactivity results (i.e. more potentGroEL/ES inhibitors with low inhibition of the native MDH and Rhoreporter enzymes). Biochemical Assay IC₅₀ (μM) Native GroEL/ GroEL/NfsB- Native Native MDH ES- ES- GroEL/ R Rho MDH Activity dRho dMDHES-dMDH GroEL Structure Group # Activity Activity w/NfsB RefoldingRefolding Refolding ATPase

Nitroxoline    2.0 >63 >56      6.4   50  27 >250

OCH₃ H  1  5  98  95 >63 >63 >56 >56   31  176   77 >100  66 >83 >250>250

OCH₃ H 29 30  66  97 >63 >63 >56 >56  117  216 >100 >100 >83 >83 >250>250

OCH₃ H 31 32 >100 >100 >63 >63 >56 >56 >250 >250 >100 >100 >83 >83 >250>250

OCH₃ H 33 34 >100 >100 >63 >63 >56 >56  129  209 >100 >100 >83 >83 >250>250

OCH₃ H 35 36 >100 >100 >63 >63 >56 >56 >250 >250 >100 >100 >83 >83 >250>250

OCH₃ H 37 38 >100 >100 >63 >63 >56 >56 >250 >250 >100 >100 >83 >83 >250>250

Nitrofurantoin >100 >63 >56 >250 >100  84 >250

Nifuroxazide  69 >63 >56  133 >100  19 >250

20 >100 >63 >56  108   69  10 >250

39 >100 >63 >56 >250 >100 >83 >250

40  18 >63 >56   30 >100 >83 >250

41  32 >63 >56   31   78 >83 >250

42 >100 >63 >56 >250 >100 >83 >250

Example 78

Evaluating the cytotoxicity of analogs to human colon and smallintestinal cells. While in previous studies we have employed biochemicalcounter-screening with the human mitochondrial HSP60/10 chaperoninsystem, our accumulating results indicate that inhibiting HSP60/10 invitro is a poor indicator of potential off-target toxicity to humancells. This is highlighted by the fact that we have identified manyknown drugs and natural products that are potent inhibitors of HSP60/10biochemical function in vitro, yet exhibit little to no adverse effectsin cells or animals. For instance, we found that suramin is a potentHSP60/10 inhibitor, yet it has been used safely for over 100 years as afirst-line treatment for Trypanosoma brucei infections. In addition, asnow identified in this study, bioactivation of nitrofuran antibiotics bynitroreductase enzymes greatly increases the extent of inhibitionagainst GroEL/ES refolding activity, and potentially human HSP60/10;however, this further complicates testing against HSP60/10 since humancells do not contain nitroreductases. Therefore, we feel the mostappropriate initial assessment of potential in vivo toxicity is to testcompounds for cytotoxicity to human cells in culture.

Example 79

Referring to FIGS. 3A, 3B, and 3C, correlation plots examining theselectivity of compounds inhibiting the proliferation of E. faecium(FIG. 3A), S. aureus (FIG. 3B), and E. coli (FIG. 3C) over cytotoxicityto human FHs 74 Int small intestine cells (results for cytotoxicity tohuman FHC colon cells are similar, with CC₅₀ values reported in Tables5, 6, and 7 and FIGS. 7A, 7B, and 7C. Results plotted in the grey zonesrepresent EC₅₀, and CC₅₀ values higher than the maximum concentrationslisted. Data points for nifuroxazide (Nfz), nitrofurantoin (Nft), andnitroxoline (Nox) are labelled for comparison.

TABLE 5 E/CC₅₀. Bacterial proliferation and human cell viability assayEC₅₀ & CC₅₀ results for hydroxyquinoline analogs 1-15. Darker grey cellshading indicates compounds with more desirable bioactivity results(i.e. more potent inhibitors of bacterial proliferation with lowcytotoxicity to human cells). Bacterial Proliferation EC₅₀ or Human CellViability CC₅₀ (μM) Colon Intestine Structure # E. faecium S. aureus K.pneumoniae A. baumannii P. aeruginosa E. cloacae E. coli (FHC) (FHS74Int)

2 >100 37 >100 >100 >100 >100 80 >100 >100

3 >100 63 99 74 >100 92 77 85 86

4 70 84 >100 >100 >100 >100 >100 25 42

5 >100 69 82 56 >100 80 63 74 57

6 >100 >100 >100 >100 >100 >100 79 >100 75

7 >100 48 87 71 >100 97 46 >100 67

1 >100 87 85 >100 95 100 >100 29 61

8 >100 99 >100 >100 >100 >100 93 35 42

9 >100 56 >100 >100 >100 >100 >100 46 60

10 >100 >100 >100 >100 81 >100 >100 71 87

11 >100 >100 >100 >100 >100 >100 >100 41 21

12 >100 >100 >100 >100 85 >100 99 34 69

13 >100 >100 >100 >100 >100 >100 >100 24 6.1

14 >100 >100 >100 84 >100 >100 >100 59 91

15 >100 >100 >100 >100 >100 >100 >100 52 40

TABLE 6 E/CC₅₀. Bacterial proliferation and human cell viability assayEC₅₀ & CC₅₀ results for nitrofuran analogs 16-28 plus nitrofurantoin andnifuroxazide. Darker grey cell shading indicates compounds with moredesirable bioactivity results (i.e. more potent inhibitors of bacterialproliferation with low cytotoxicity to human cells). BacterialProliferation EC₅₀ or Human Cell Viability CC₅₀ (μM) E. S. K. A. P.Colon Intestine Structure # faecium aureus pneumoniae baumanniiaeruginosa E. cloacae E. coli (FHC) (FHS 74Int)

Nitrofurantoin 38 27 40 >100 >100 36 0.69 >100 >100

16 24 7.1 16 82 >100 40 0.42 83 71

17 12 3.0 19 72 >100 49 0.45 53 85

18 31 10 36 >100 >100 >100 2.1 >100 94

19 13 9.5 42 54 >100 73 2.4 61 90

Nifuroxazide 8.1 16 37 >100 >100 54 0.87 61 79

20 22 8.1 >100 >100 >100 >100 1.9 >100 75

21 >100 34 >100 >100 >100 >100 10.0 >100 93

22 16 6.0 >100 >100 >100 >100 41 67 87

23 11 5.2 >100 >100 >100 >100 >100 47 80

24 11 3.5 >100 >100 >100 >100 >100 54 72

25 >100 9.6 >100 >100 >100 >100 >100 >100 >100

26 11 11 >100 >100 >100 >100 17 >100 70

27 >100 >100 >100 >100 >100 >100 >100 90 >100

28 >100 >100 >100 >100 >100 >100 >100 85 >100

TABLE 7 E/CC₅₀. Bacterial proliferation and human cell viability assayEC₅₀ & CC₅₀ results for additional analogs examining the substructuresof the hydroxyquinoline and nitrofuran aryls that are important forinhibition. Darker grey cell shading indicates compounds with moredesirable bioactivity results (i.e. more potent inhibitors of bacterialproliferation with low cytotoxicity to human cells). BacterialProliferation EC₅₀ or Human Cell Viability CC₅₀ (μM) K. Intestine R E.S. pneu- A. P. E. E. Colon (FHS Structure Group # faecium aureus moniaebaumannii aeruginosa cloacae coli (FHC) 74Int)

Nitro- xoline  18     9.5     2.8     2.5  99     6.6     4.0  28  41

OCH₃ H  1  5 >100 >100  87  69  85  82 >100  56  95 >100  100  80 >100 63  29  74  61  57

OCH₃ H 29 30 >100 >100 >100 88 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100>100

OCH₃ H 3132 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100>100

OCH₃ H 3334 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100>100

OCH₃ H 3536 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100>100

OCH₃ H 3738 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100>100

Nitro- furantoin  38  27  40 >100 >100  36      0.69 >100 >100

Nifur- oxazide     8.1  16  37 >100 >100  54      0.87  61  79

20  22     8.1 >100 >100 >100 >100     1.9 >100  75

39 >100 >100 >100 >100 >100 >100 >100 >100 >100

40     7.3  19  15  11 >100  24  14     4.4  15

41  20     5.8  10  12 >100  13     9.5  53  63

42 >100 >100 >100 >100 >100 >100 >100 >100 >100

Example 80

To assess for potential cytotoxic effects, compounds were tested in twoAlamar Blue-based cell viability assays using human FHC colon and FHs 74Int small intestinal cells. Briefly, we grew cells to ˜80-90%confluency, then sub-cultured 1,500 cells per well (in 384-well plates)for 24 h in the absence of test compounds. Compounds were then added andthe cultures were incubated for an additional 48 h, whereupon the AlamarBlue reporter reagents were added and well fluorescence was monitoredover time. Alamar Blue contains resazurin (non-fluorescent), which isreduced to resorufin (highly fluorescent) in the presence of viablecells. Cell cytotoxicity CC₅₀ values in are shown Tables 5, 6, and 7. Asgraphically presented in FIGS. 3A, 3B, and 3C correlation plotscomparing bacterial proliferation EC₅₀ to human cell cytotoxicity CC₅₀results, lead nitrofuran inhibitors (16, 17, 20, nitrofurantoin, andnifuroxazide) selectively inhibited E. faecium, S. aureus, and E. coliproliferation with low to no cytotoxicity to human cells (representativeresults are shown for human FHs 74 Int small intestine cells, butresults are similar for FHC colon cells and are presented in FIGS. 7A,7B, and 7C). Intriguingly, the nitrofuran analogs were typically lesstoxic than their hydroxyquinoline counterparts, putatively because theywould need to be metabolized to their active intermediates, yet humancells do not harbor nitroreductases.

Example 81

Investigating the ability of E. coli to gain resistance to 17,nifuroxazide, and nitrofurantoin. As we discovered severalnitrofuran-based analogs that selectively inhibited the growth of E.faecium, S. aureus, and E. coli with minimal toxicity to human cells, wenext investigated how easy it would be for bacteria to generateresistance to a lead candidate. We examined the ability of E. coli togenerate resistance to 17 (with nifuroxazide and nitrofurantoin ascontrols), since resistance to nitrofuran-based antibiotics has beenwell-characterized in this bacterium. While 17, nifuroxazide, andnitrofurantoin were all potent inhibitors of E. coli proliferation, 17was the most potent at inhibiting GroEL/ES in the presence of NfsB, andthus may exhibit greater on-target effects in bacteria. However, asdiscussed above, we do appreciate the limitations of not employing NfsA.To identify differences in the ability of E. coli to generate resistanceto these three compounds with distinct bioactivity profiles, we employeda 12 day resistance assay as we previously reported for ourhydroxybiphenylamide lead candidate S. aureus inhibitors. A diluteculture of E. coli (OD₆₀₀=0.01) was grown in the presence of inhibitorsfor 24 h (tested in dose-response in duplicates), then EC₅₀ results weredetermined from the OD₆₀₀ readings of the wells. Over the course of 12days, we sequentially sub-cultured bacteria from the respective wellswith the highest concentration of inhibitors where bacteria grew to anOD₆₀₀ >0.2, monitoring for increases in EC₅₀ values over time.

Example 82

While we found that nifuroxazide and nitrofurantoin were initially morepotent than 17 as shown in FIGS. 4A, 4B, and 4C, E. coli quicklydeveloped intermediate resistance (within 3-5 days) such that all threeinhibitors were nearly equipotent. This initial resistance wasputatively through mutations affecting NfsA function, as previouslyreported. That EC₅₀ values then somewhat plateaued in the 20-40 μM rangeis consistent with NfsB still being able to metabolize the nitrofuranmoieties and maintain efficacy, albeit at a reduced capacity. EC₅₀values continued to slowly increase over time for all three compounds,with a particular jump in resistance seen for the second set ofreplicates for nitrofurantoin and nifuroxazide to a lesser extent, butnot for 17. Thus, inhibitors that are preferentially activated by NfsB,as may be the case for 17, might be more effective drug candidates withrespect to combatting the emergence of drug resistant strains.

Example 83

Referring to FIGS. 4A, 4B, and 4C, evaluating the ability of E. coli togenerate resistance to nifuroxazide (FIG. 4A), nitrofurantoin (FIG. 4B),and compound 17 (FIG. 4C) over time. Time-course plots show the changein EC₅₀ values for each compound over the 12-day serial passageresistance assay (compounds tested in duplicates, as indicated by theblack and white triangles).

Referring to FIGS. 5A, 5B, and 5C, dose-response curves for nifuroxazide(FIG. 5A), nitrofurantoin (FIG. 5B), and compound 17 (FIG. 5C) testedagainst the susceptible parent E. coli (white triangle), themaximally-resistant strains developed to the respective test compounds(black triangle), and follow-up proliferation assays for resistantstrains tested after serial passaging in the absence of test compoundsto account for possible reversible inhibition mechanisms (greytriangles). Results are presented for the replicate 1 samples from theresistance assay, with results for replicate 2 samples similar andpresented in FIGS. 8A, 8B, and 8C. In all instances, the resistantstrains were nearly equally resistant even after culturing in theabsence of inhibitors for the 4×12 h passages.

Example 84

We next confirmed that the resistance generated by the replicate E. colistrains was irreversible (i.e. putatively through permanent mutations ofNfsA and NfsB as previously reported) as opposed to transient means(i.e. by up-regulating efflux pumps). To accomplish this, wesub-cultured single colonies obtained from the replicate samples wherethe bacteria exhibited the greatest degree of resistance to testcompounds (day 12 samples for all compound replicates, except replicate2 for nitrofurantoin, which was taken at day 10), for 4×12 h serialpassages in fresh media without any test compounds present. We thenperformed another 24 h follow-up proliferation assay to determine EC₅₀values (dose-response curves are presented for nifuroxazide,nitrofurantoin, and 17 in FIGS. 5A, 5B, and 5C and FIGS. 8A, 8B, and 8C.Results indicated that these subsequently-cultured bacterial strainswere still resistant to each of the respective inhibitors they weregenerated from, supporting that resistance mechanisms were permanent. Asprevious studies by others have extensively characterized mutationsaffecting NfsA and NfsB that E. coli acquire to generate resistanceagainst nitrofuran antibiotics, we did not perform genotyping to furthercharacterize the specific resistance mechanisms for these strains, asthey were likely the same.

Example 85

Since nifuroxazide, nitrofurantoin, and 17 displayed differentinhibition and resistance profiles, we next examined if the respectiveresistant strains were cross-resistant with each of the otherinhibitors. While the replicate strains that were initially resistant tonifuroxazide were cross-resistant to both nitrofurantoin and 17 (SeeFIGS. 6A and 9A), the nitrofurantoin-resistant strains were stillsensitive to nifuroxazide and 17 (FIGS. 6B and 9B), and the 17-resistantstrains were susceptible to nifuroxazide, but not nitrofurantoin (FIGS.6C and 9C). Thus, it is evident that strains that have generatedresistance to one analog are not necessarily cross-resistant to otheranalogs.

Example 86

Referring to FIGS. 6A, 6B, and 6C, evaluation of cross-resistancebetween the respective resistant E. coli strains with nifuroxazide,nitrofurantoin, and compound 17. The three panels show dose-responsecurves for the three inhibitors tested against strains where resistancewas initially generated to nifuroxazide (FIG. 6A), nitrofurantoin (FIG.6B), and compound 17 (FIG. 6C). Results are presented for the replicate1 samples from the resistance assay, with results for replicate 2samples similar and presented in FIGS. 9A, 9B, and 9C. Results indicatethat resistance generated to one inhibitor is not necessarilycross-resistant to the other inhibitors, potentially indicatingdifferent mechanisms of activation and/or targets.

Example 87

In a previous high-throughput screen, compound 1 was identified as amoderate GroEL/ES inhibitor with weak to moderate antibacterial efficacyagainst B. subtilis, MRSA, K. pneumonia, A. baumannii, and SM101 E. coli(which has a temperature sensitive LPS biosynthetic pathway). Keysubstructures of compound 1 resembled those of nitroxoline (i.e. sharedhydroxyquinoline moiety) and nitrofuran-based antibacterials such asnifuroxazide and nitrofurantoin (i.e. shared bis-cyclic-N-acylhydrazonecores). Thus, we developed two parallel series of hydroxyquinoline andnitrofuran-bearing compound 1 analogs in an effort to increase inhibitorpotency against GroEL/ES and E. coli and the ESKAPE bacteria, whilereducing cytotoxicity to human cells (a compilation of assay results fornitrofurantoin, nifuroxazide, and lead analogs 16-21 is presented inTable 8). Initially, only the hydroxyquinoline series was found tocontain potent GroEL/ES inhibitors in our traditional GroEL/ES-mediatedsubstrate refolding assays; however, subsequent testing in the presenceof E. coli NfsB indicated that the nitrofurans act as pro-drugs andtheir reactive metabolites can be potent GroEL/ES inhibitors. Leadnitrofuran analogs were potent inhibitors of E. faecium, S. aureus, andE. coli proliferation and exhibited minimal cytotoxicity to human FHCcolon and FHs 74 Int small intestinal cells. While E. coli were able togenerate varying degrees of irreversible resistance to nifuroxazide,nitrofurantoin, and lead analog 17 (putatively through mutations in NfsAand NfsB), clones were not necessarily cross-resistant to the otherinhibitors. This finding may indicate diverging mechanisms of activationand/or targets for structurally distinct inhibitors, suggesting thatcombination therapy could prove synergistic and delay the onset ofbacterial resistance.

TABLE 8 Cell-based and biochemical EC₅₀, CC₅₀, and IC₅₀ results for thetop eight lead inhibitors based on average Selectivity Indices (SI) forinhibiting E. coli proliferation over cytotoxicity to human colon andintestine cells. For the GroEL/ES-dMDH refolding assay and native MDHcounter-screens, IC₅₀ results are shown for compounds tested in theabsence and presence of NfsB nitroreductase (w/ and w/o NR,respectively). Cell-Based Assay EC₅₀ & CC₅₀ (μM) Biochemical Assay IC₅₀(μM) Bacterial Proliferation Human Cell Native MDH GroEL/ES-dMDHrefolding: E. Viability SI (CC₅₀/EC₅₀) w/o w/ w/o w/ Compound Structure& Name/Number E S K A P E coli Colon Intestine Colon Intestine NR NR NRNR

16 24 7.1 16 82 >100 40 0.42 83 71 198 168 >63 >56 >100 7.7

17 12 3.0 19 72 >100 49 0.45 53 85 119 190 >63 >56 88 3.2

Nitrofurantoin 38 27 40 >100 >100 360.69 >100 >100 >146 >146 >63 >56 >100 84

Nifuroxazide 8.1 16 37 >100 >100 54 0.87 61 79 70 91 >63 >56 >100 19

20 22 8.1 >100 >100 >100 >100 1.9 >100 75 >53 40 >63 >56 69 10

18 31 10 36 >100 >100 >100 2.1 >100 94 >48 45 >63 >56 >100 12

19 13 9.5 42 54 >100 73 2.4 61 90 26 38 >63 >56 >100 23

21 >100 34 >100 >100 >100 >100 10.0 >100 93 >10 9.3 >63 >56 26 12

What is claimed is:
 1. A compound of formula (I):

wherein R¹ is C₆-C₁₀ aryl, biaryl, or mono- or bicyclic heteroaryl,wherein each hydrogen atom in C₆-C₁₀ aryl, biaryl, or mono- or bicyclicheteroaryl is optionally substituted with halogen, —OR⁵, —NR⁵R⁶,—S(O)₂NR⁵R⁶, or —N(R⁵)SO₂R⁶; R² is hydrogen; or R¹ and R² combine withthe atoms to which they are attached to form a 3- to 7-memberedheterocycloalkyl optionally substituted by oxo; R³ is C₆-C₁₀ aryl,biaryl, or mono- or bicyclic heteroaryl, wherein each hydrogen atom inC₆-C₁₀ aryl aryl, biaryl, and mono- or bicyclic heteroaryl is optionallysubstituted by —OR⁵ or nitro; R⁴ is hydrogen; R⁵ and R⁶ are eachindividually hydrogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl, wherein each hydrogen atom in C₁-C₆ alkyl, C₆-C₁₀ aryl, andmono- or bicyclic heteroaryl is optionally substituted with halogen or—OC₁-C₆ alkyl; or a pharmaceutically acceptable salt thereof, providedthe compound is not


2. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R³ is mono- or bicyclic heteroaryl optionallysubstituted by nitro.
 3. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R³ is quinolyl optionally substitutedby —OR⁵.
 4. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R³ is napthyl optionally substituted by —OR⁵. 5.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R³ is furanyl optionally substituted by nitro.
 6. The compoundof claim 1, having the formula (II)

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, having the formula (III)

wherein Z is CH or N; or a pharmaceutically acceptable salt thereof. 8.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is monocylic heteroaryl optionally substituted with halogen.9. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is C₆-C₁₀ aryl optionally substituted with halo,—OR⁵, —NR⁵R⁶, —S(O)₂NR⁵R⁶, or —N(R⁵)SO₂R⁶.
 10. The compound of claim 7,wherein R¹ is C₆-C₁₀ aryl optionally substituted by —OH, —OC₁-C₆ alkyl,O-aryl, —N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —N(H)S(O)₂—C₆-C₁₀ aryl,—N(H)S(O)₂-monocylic heteroaryl, or —N(H)S(O)₂-bicylic heteroaryl,wherein each hydrogen atom in C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl is optionally substituted with halogen or —OC₁-C₆ alkyl. 11.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is biaryl.
 12. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, selected from the group consisting of


13. The compound, of claim 10, or a pharmaceutically acceptable saltthereof, selected from the group consisting of


14. The compound of claim 11, or a pharmaceutically acceptable saltthereof, selected from the group consisting of


15. The compound of claim 10, or a pharmaceutically acceptable saltthereof, selected from the group consisting of


16. The compound of claim 13, or a pharmaceutically acceptable saltthereof, selected from the group consisting of


17. A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and optionally at least onediluent, carrier or excipient.
 18. A method of treating a bacterialinfection comprising administering to a subject in need of suchtreatment an effective amount of at least one compound of claim 1, or apharmaceutically acceptable salt thereof.